JPS60115529A - Non-glycosylated amino acid chain immunologically reactive to glycoprotein of herpes virus 1 and 2 - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

The present invention relates to a vaccine for the prophylaxis and treatment of herpes simplex virus types 1 and 2 (commonly abbreviated as H8V-1 and H8V-2). More specifically, nucleotides excluding genes other than those necessary for expressing an amino acid chain that is composed of all elements having a sequence matching the amino acid chain found in glycoprotein B of H8V-1 and H8V-2. It relates to the application of recombinant, recombinant DNA technology for the production of plasmids containing the amino acid chains expressed by the DNA, and those that produce an effective immune response against H8V-1 or H8V-2. derivatives, and vaccines based on the amino acid chain products produced by their DNA, respectively. The present invention relates to living prokaryotic cells, living eukaryotic cells (which have the ability to insert and extract recombinant DNA, and are capable of expressing the DNA molecules).
A DNA molecule used to direct the synthesis of the protein portion in glycoprotein B is provided. Recombinant D of the present invention
Using NA technology is cost effective and the amino acid chains produced are synthetic, prophylactic and therapeutic agents without the risk of contamination with live viruses. The DNA of herpes simplex virus types 1 and 2 is 15
It contains 0 kb and has enough information to encode 100-200 proteins. Approximately 30 proteins are present in the virus particle, and the remaining encoded proteins are involved in the replication and assembly of the virus in the host cell. In addition to a DNA core surrounded by a protein membrane, viruses have an outer membrane structure. The membrane is composed of lipids and virus-encoded glycoproteins. These glycoproteins are proteins with one or more sugar chains that begin with unique amino acid residues. 4 H8
V glycoprotein gA/gB (gA is the precursor of gB),
There are gC, gD, and gE. H8V probably enters host cells by fusion of the viral and cellular membranes, and this lysis is mediated by gB. Viruses replicate in the nucleus. During replication, glycoproteins are synthesized and transported to the cell surface, with many of the glycoproteins reaching the outer plasma membrane surface of infected cells. Viral glycoproteins are the first viral molecules that bind to H8V-specific antibodies as they exit from the viral and cell surfaces. Vaccines generally consist of virus particles (
It is made up of live viruses, attenuated viruses, modified viruses] or structural proteins of viruses (subunit vaccines).Although there are reports confirming the effectiveness of live H8V vaccines, many experts do not recommend the method. Not recommended.Why in Europe?
This is because it is not a type of virus that threatens human survival. In contrast, subunit vaccines are composed of one or more glycoproteins or their non-glycosylated (non-glycosylated) amino acid chains, and either immunologically active site is preferred. . Live vaccines are attractive because they are cheap to produce, and because they multiply after vaccination, very small quantities are required. However, it is potentially dangerous. Subunit vaccines require the production of large amounts of protein and are expensive, but they are safe. The viral glycoprotein and protein components synthesized in E. coli by recombinant DNA technology according to the invention are completely free from viral DNA constraints. There is only one viral glycoprotein gene, not DNA with all the elements. Recombinant DNA technology eliminates the risks associated with live virus contamination in vaccine production. Only one of the major viral proteins, gB, has a known biological function and has been shown to be a fundamental component of the virus growth process. The table following the example below shows the D'NA coding sequences of gB each of H8V-1 and H8V-2. The present invention provides glycoprotein B
shows a nucleotide sequence capable of expressing an amino acid chain of By inserting a gene into a microorganism, a protein corresponding to the H8V glycoprotein can be synthesized. Although only the protein part, and not the sugar part, of gB is synthesized in bacteria, the protein is the major source of immune determinants. For expression, a microorganism, typically a bacterium such as E. coli, is grown in a conventional growth medium containing a plasmid capable of expressing the required amino acid chain. In the present invention, purified H8V proteins of the present invention, extracted from bacterial membrane fragments, can be inoculated into animals (eg, rabbits) to generate antibodies against the proteins. These antibodies produced within the animal body in response to the gB protein produced in bacteria can be tested for their ability to inactivate (neutralize) the virus. After H8V-gB expression was maximally increased in bacteria and after demonstration of adequate virus neutralization by antibodies generated against gB, the gB protein vaccine was tested in animals and finally in clinical trials. can be evaluated in A single inoculation of approximately 3 μg protein/9 to rabbits will generate an antibody response. Although the complete encoded sequence of gB could be expressed in bacteria, there are practical reasons why it is desirable to express only the immunologically active portion of gB. If the three-dimensional structure of gB expressed in the E. coli cytoplasm could be altered, there would be two extensive hydrophobic sites, a signal site and a membrane bridging site. (See Summary of Sequences below.) When the completed protein is present in the membrane of an infected cell, the signal sequence is enzymatically removed and the bridging residues to the membrane are present in the membrane. The remaining C-terminal residues on the cytoplasmic side of the membrane are inaccessible to the antibody. Therefore, it appears that up to the 75ON terminal amino acid residues of the entire 903 are antigenically active with respect to the vaccine. Hydrophilic residues appear to be present on the protein surface. These residues in the region of the β-helix are candidates for antigenic determinants. However, since the primary structure and the tertiary structure are not one-dimensionally related, it cannot be said that the hydrophilic residues forming the antigen site are necessarily close to each other. Nevertheless, a suitably selected of the long chains, with a length of 10-20
Adjacent amino acid residues are often antigenic and can lead to the production of antibodies that bind to the native protein that contains that residue. Therefore, the simplest vaccine would be a short peptide with high antigenic activity, the sequence of which is derived from knowledge of the nucleotide sequence of the gB gene. Representative recombinant DNA clones of herpes simplex virus type 1 in the present invention, exemplified by a clone called H8V-1 plasmid pKBXX, are constructed by the method shown below. DNA purified from the KO8 strain is cleaved using the restriction endonuclease Bam HI using reaction conditions determined by the restriction enzyme supplier. At the same time, the vector DNApBR322 (see Boljvar et al.
al, e Gene 2.95-113.1977]
is cut using gam HI. Kernel enzymes can be removed and inactivated by extraction with organic solvents. Then, the DNA is precipitated with ethanol at low temperature and the remaining solvent is removed by drying by suction. BamHI cleaved H8V-I DNA
A truncated pBR322 vector (this vector contains the tetracycline resistance gene (JLL unique in rerl)
ligated with the similar viscous end of the 3-III HI site). Ligation is conveniently performed using T 4 DNA ligase at about 4° C. according to conditions provided by the manufacturer. Both the cleavage and conjugation reactions are carried out in sealed plastic containers. A similar continuation of the reaction was carried out using the KO8 strain again, cutting with the same number of enzyme units of the restriction endonuclease EcoRI and then cutting the vector DNA pBR using coRI.
325 (Bolivar, Gene 4. 121-136:1978). The enzyme was again removed and the EeORI-cut H8V-1 was cloned into pBR325 (a unique Ec
It was ligated to the similar end of J with an oRI site. Plasmid pBR325 contains ampicillin (Apr),
It contains genes that clearly show drug resistance to tetracycline and chloramphenicol, and pBR322 has genes for ampicillin resistance and tetracycline resistance. As a result of the introduction of the ligation mixture into E. coli, vectors presumed to contain H8V-1 fragments were treated with chloramphenicol for pBR325 and tetracycline for pBR322 (which loses growth ability in the presence of these substances). It is identified by resistance to ampicillin rather than susceptibility to Drug resistance genes and other genes are almost always inactivated by foreign fragments or extraneous DNA. Recombinant plasmids containing the tmHI-F fragment or the μtmHI-G fragment are H8V-1 for these enzymes.
is identified by comparing the electrophoretic mobility of the H8V-1 insert with the known restriction map of H8V-1. Restriction fragments are designated by capital letters A-Z in order of increasing mobility on the agarose gel, consistent with decreasing nucleotide length. H8V-1 homologous strain 1 or K
O8 will represent a unique restriction map for any one restriction endonuclease. but,
Different strains of H8V-1 propagate completely independently of KO8 and therefore share most, if not all, of the same restriction sites. For subclone (5ubclone identification)
(Especially the E obtained from the KO8 and ts135 strains)
colRI-F fragment] KOS (7) The coordinates (coodjnate) for the EcoRI-F fragment are used. The following restriction enzymes are marked in the restriction map for the subclone of the EcoRI-F fragment of the H8V-1hKO8 strain. The parts are shown below. ￥ ω ω mountain kXE (1') V3 cQ O('-1004C'J(v'
) Dimension 0 〇- Mountain a: lkl+kA-+? CS3 dimension-■ωto-eP dimension [F]■- m ct'z CQ l-Q cn dimension 0 0 0
() OOO l:! :l ω ooH< mountain mountain × ni)0 0
() u 00 0) (Th to C'J ω dimension dimension Q ■ O to Kakuyama to m ω li −× ■0■00to's O I+ ω ω dimension Oω to O% fEEL “92” KO8 system and temperature Comparison of restriction endonuclease cleavage site maps for susceptible tsB5 strains is 0.31
to 8: 0.344P; 0.355T; and 0.413
Showing specificity for KO8 at the site of T, 0.33
1) I, 0.355P, and 0.389X. Shows specificity for tsB5 at the site. A comparison between the restriction map for the EcoRI-F fragment and that of the tBB5 strain shows that the two strains have genomic coordinates 0
．． 315 to 0.422, all but 7 sites in the EcoRI-F fragment are shared. The H8V-1 genome is a 150 kb linear double-stranded DNA.
It is the A molecule. Therefore, 0.01 map unit is approximately 1.5
It corresponds to kb. The H8V-1gB pkBXX plasmid consists of a structure containing H8V-1 sequences from 0.345 BamHI to 0.372Xhor. 0.345
JiH1-G with coordinates from part to 0.399 part
Fragments are first made by the ligation method described above and then 5
It undergoes partial digestion with alI and is ligated again to remove the SalI fragment. This yields a fragment pKBG-BS3 with coordinates from the 0345 digit H1 site to the 0.3878 al1 site. This is two Xho
lo, 368 and Q, 372) sites. In pKBXX, to obtain the requested sequence, p
Complete digestion using KBG-BS3 and HI and Xho
undergo partial digestion using l. As a result of agarose electrophoresis, 13amHI from 0.345 to 0.372
-Xho I fragment and corresponding 4, Okb bound binding layer, electroeluted from the gel, gam old, reciprocal I cleaved p
It is ligated to UC9. pUC9 is a 2.7kb protein containing the ampicillin resistance gene and multiple cloning sites.
It is a plasmid. (See, Vieira & Mes.
sing, Genel 9:259-268°198
2; Messing & Vieira *Same book:
2691゜This plasmid was purchased from Bethesda Research Labs,
) available from The origin of replication of the plasmid and ampicillin resistance gene is pBR322 (derived approximately from nucleotides 2067 to 43621. pU
The remaining sequence of C9 (approximately 400 nucleotides) is derived from the early stages of the E. coli carrier gene. In particular, the sequence includes a facilitator for RNA synthesis, a liposome binding site for protein synthesis, and an N-terminal fragment of the first gene product (β-galactosidase). The multiple cloning site is the restriction site H4ndIII-PstI-5alI- inserted between amino acids 4 and 5 of the characteristic sequence of E. coli β-galactosidase.
BamHI - SmaI = EcoRI I (from left to right). (See Table 3) It has been shown that β-caractosidase protein synthesis is not affected by the amino acid sequence near its N-terminus (as long as the correct reading frame is maintained). Galactosidase is pUC9
Synthesized in cells containing plasmid DNA. A vector with a unique restriction site closely associated with the H8V-1 cloned sequence (but not present in the H8V-1 sequence) allows the H8V-1 clone to be recloned into several other vectors with the same restriction site. pKB
To create XX, pUC9 DNA is treated with BamHI and 5alI and the H8V-1 sequence (BamHI to XhoI f 0.345 to 0.372) is ligated to these treated sites. The appropriate loans (after transformation analysis) were identified by restriction mapping. The H8V-1 sequence contained within pKBXX was shown to be among the restriction site descriptions for obtaining the restriction map described above. 5alI [G TCGA C) and XhoI fc
Since the recognition site of TCGA C1 differs depending on the state of the terminal nucleotide site, they cut DNA at different sequence sites, but the highly viscous terminal site + TCGA1
It should be noted that the same four bases are produced. Therefore, the Xhol site is cloned into the 3al I site and the sequence is made in pKBXX. H8
The base sequence at the V-IXhoI-pBR322SalI junction becomes the hybridization site CTCGAC. This part is
Not cleaved by either enzyme used to produce the recombinant plasmid. Nucleotides are numbered from 1 to 3997 in pKBXX from right to left (XhoI to BamHI). All nucleotides in pKBXX were extracted from the M13 cloning vector [dideoxy chain-end method.
ain-termination method
]. A summary of the sequence by nucleotide number is shown below. From Sequence Summary 1 to approximately 375 5' extra sequences starting at the 0.372 Xho I site (5
'extra 5 sequences 1 approx. 375
two CAA starting at nucleotides 406 and 443 from to 499
5' containing T-box and TATA-box at 476
mRNA regulatory sequences. m RNA A starting sequence from 499 to 789 reaching the first encoded nucleotide starting with nucleotide ≠ 790 (the first A is ≠ 501
Starting with A of CaCCaCAC, from 790 to 3498, there is an N-terminal hydrophobic leader, a membrane bridging sequence, a C-terminal ionic sequence, and an amino acid coding sequence for sugar addition. The region of the glycoprotein that will be present outside the cell surface is a 75-amino acid chain. 3' non-coding sequence containing the 3518 polyA addition signal start site and the extant 3549 polyA addition site from 3499 to 3546. 3992 BamHI sites from 3549 to 3997 (coordinates teordinat
e) 3' non-essential sequence to 0.345). Table 1 shows the complete nucleonate sequence of pKBXX, and Table 2 shows the complete translation product. The important peptide composition is N
contains a terminal end (extensive hydrophobic membrane insertion sequence or isohydrophobic signal sequence), nine viable N-cyclic glycosylation sites, and a high charge anchoring sequence (109 amino acids). is excluded by specific signal peptidases during glycoprotein synthesis to generate the complete peptide. H8V-2 plasmid H8V-2 shows gene homology with the gB gene of H8V-1. A typical recombinant DNA clone of herpes simplex virus type 2 in the present invention is created by the method shown below.The DNA purified from the HG52 strain is digested with restriction enzymes. The vector DNApBR325 is cleaved using the restriction endonuclease EcoRI using reaction conditions determined by the supplier.
Disconnected using RI. This enzyme can be removed and inactivated by extraction with organic solvents. The DNA is precipitated with ethanol at low temperature and dried by suction to remove residual solvent. The KatsuRI-cut H8V-2 DNA is ligated to the same sticky ends of the EcoRI-cut pBR325 vector (which has a unique EcoR1 site in the chloramphenicol resistance gene). Ligation is efficiently performed using T4 DNA licase at approximately 4° C. according to conditions provided by the manufacturer. Plasmid pBR325 contains genes showing drug resistance to ampicillin, chloramphenicol, and tetracycline. After insertion of the ligation mixture into E. coli, H
Cells containing vectors presumed to contain the 8V-2 fragment are resistant to ampicillin (or tetracycline) and sensitive to chloramphenicol (unable to grow in the presence of chloramphenicol). Identified. The drug side genes and other il'r genes are almost always inactivated by the insertion of large heterologous DNA fragments. Groups (colonies) of chloramphenicol-sensitive clones
A selection is made to determine which clones contain the gB sequence. To do so, isolated bacterial colonies were regrown in special mouthpieces placed on top of a dish containing solid growth medium. Colonies obtained by sniffing were fixed in places, and further treated with NaOH to remove externally added D.
Becomes to penetrate NA. This treatment also allows double-stranded D
Separate NA into single strands. A solution of radioactive-defective DNA derived from the coding sequence of H3V-1gB was poured onto the mouthpiece, which was incubated and washed to the septum with the appropriate buffer. H3V-2 containing plasmid DNA in special colonies becomes H3V-1,!
: With homologous (basically identical) nucleotide sequences, the nucleotide sequence of HSV-1 will be uniquely fixed and retained on the stomach. This process is called hyfrit formation, in which the double-stranded DNA held in the stent is combined with one HSV-1 radioactive strand.
meant to include the H8V-2 non-radioactive chain. The opening paper is transferred to X-ray film, and the hybridization is
The desired colony is detected by creating a dark spot at that location. By this method, Coroni has p52
It was confirmed that the vector contained an 11 SV-2 Eco RI fragment called EG. Two colonies showed extreme hybridization and contained inserts. The mobility of the insert was indistinguishable from that of EcoRI-G. The insert also contains a unique Hindl11 site characteristic of the EcoRI-G fragment (as determined by comparison with the HSV-I EcoRI-F fragment, which appears to share the same coordinates). The first clone f p52EG) was +3 a m H1 or Hi
ndlll was cut using T, and the cut J) was cloned into the same site of pUC9 after cell elution. It is called p52EG-BH. Vector p particularly suitable for DNA expression into bacteria-c1 protein
It has been discovered that it is advantageous to clone clones derived from p52EG into UC9. Smaller plasmids include complete BamHI and Hindl
ll digestion, then N rib I + 0.354 and 0.372
p52E
Made from C-DH. Appropriate gel-eluted fragments (BamH
I-XhoI-XhoIt or BXX) was subsequently cloned into the upper trunk and 5alI site of the second pUC9 vector creating a 0.372 Sal I-XhoI hybridization site. The coordinates of the insert are o345-0.37 according to the restriction map.
2, and DNA sequence analysis shows that 0.368
An area of -0.348 was eventually shown. Approximately one of the three restriction sites is common between H8V-2 and H8V-1. Those sites are used to normalize the coordinates of HG52 to similar sites in the H8V-I KO8 strain. For example, a 0.360 Sal I site and a 0.360 Sal I site.
The 348 BstE11 site is identical in the two strains. H8V-1 plasmid pKBXX (7) H8V −
2 homologs. HSV-2 derived plasmid 952
BXX is gBmRN outside the 5' and 3' ends.
It contains about 4000 base pairs with about 450 nucleotides of A. This is 0.345 proximal BamHI site to 0.372 Xho I at H8”17-1
It extends to the 0,372 Xho I site, which is within 10 bp of the site. The site is likely a site at the same nucleotide in the two genomes. The BamHI site in H8V-2 is similar to the BamHI site in H8V-1.
50 nucleotides to the right of the site. The table shown below shows the relative positions of restriction sites. H8V-H8 between 0.345 and 0.372 map units
V-1tKos l H8V-2[HG-52]Bam
HI O, 3450, 3453 B+qtEII O, 349, 0, 3550, 349X
ma I O, 347, 0, 3540, 350, 0, 3
597,0,357,0,35970,371 NruI O,3690,369 PstI O,350,0,35630,356,0,
3563,0,3680,367,0,368 XhoI O,3682,0,37160,354,0
,3716 SalI O,3600,358,0,36
0Bal I O, 3575, 0, 36750, 357
5,0,3675 Table 2 also shows H8V-2gB and H8
A comparison of DNA base and amino acid sequences for V-1gB is shown. The nucleotide and amino acid sequences of H8V-1 are shown, and some mutations in the H8V-2 sequence (H
8V-1]. H8V-1gB contains asn-pro at the persisting site at residues 76-78 in the nine NJJ-like glycosylation sites.
-thr- in H8V-2, gly -pr
Changes to o-arg. Finally, we discovered five codons that are absent in H8V-1 but present in H8V-2. This is indicated by the space in the sequence for H8V-1, and the inserted sequence for H8V-2 is written above the blank space. The H8V-2gB gene is
It shows great homology with that of 8V-1. From pKBXX, to express gB, the plasmid is cut, for example, at the NruI specific site at nucleotide 482, and for its expression a large number of 5' non-coding nucleotides and a 5' hydrophobic signal-designating nucleotide are added. In order to remove portions or portions of nucleotides 1 to 911, both Ba13115' to 3' and 3' to 5' are treated with an enzyme preparation containing an exonuclease active component. gB using an antibody that precipitates gB after religation and transformation of E. coli.
Bacterial clones can be tested for their ability to produce. Note that in pKBXX, the H8V-1 sequence was inserted at a position in the multiple coding site that corresponds to amino acids 6 to 8 of β-galactosidase (see Table 3). The gB gene is therefore fused to the β-galactosidase gene at this position. However, gB has a chain terminal codon at nucleotide 3498 (Table 2
], it is not fused to β-galactosidase at the carboxyl terminus of β-galactosidase. Although pKBXX can be used to express essentially all of the completed gB peptide, there are several reasons why it is desirable to express only the portion of gB that is antigenically active. First, large membrane proteins containing hydrophobic sequences are often unstable upon expression in bacteria. Second, approximately 75 of the total peptides
Only the ON-terminal amino acids have antigenic activity. This is because the remaining residues are located in the membrane and are therefore inaccessible to antibodies. Third, the relationship between three-dimensional structure and antigenic activity is unknown for any protein. Therefore, it is not possible to predict a single set of sequences that are most likely to have the highest antigenic activity. Therefore, a set of DNA plasmids was selected, each containing a portion of the N-terminal part of gB (beginning beyond the signal region) for the production of antigenically active gB in bacteria. One group of plasmids includes the use of a 1477-bp μl restriction piece of nucleotides extending from 637 to 2114 in both pKBXX and p52BXX. Purified fragments are incubated with Ba131 for different times to remove from each end of the fragment 0 to approximately 30" Obp. 275" from the 5' end.
By removing the nucleotide, nucleotide 91
1 or just at the xylem of the hydrophobic signal sequence, creating a new V S /terminus. After Bal 31 digestion, approx.
The length of nucleotides from 00 to 1477 bp corresponds to the N-terminal amino acids 267 to 492 of gB. A group of fragments is pooled and the plant end is
and 11 digested pUC91 plants were terminated and ligated. As a result of bacterial transformation, individual bacterial progeny are tested for production of antigenically active gB using radioimmunoassay. (See Example 7) The plasmid, which leads to the production of large amounts of antigenically active gB, is identified and its sequence determined to establish the nucleotide coordinates in the xylem of the plasmid. Such fragments, as well as fragments from the straight-through portion of the gB gene, are used to create vaccines. gB
Antibodies against the peptide can be used to determine the degree of virus neutralization. If the gB vaccine does not have sufficient antigenic activity,
Expression will require a eukaryotic vector. For this purpose, so-called shuttle vectors can be used, which vectors can be used to transport D in bacteria or in eukaryotic cells.
Duplicate NA. The endogenous promoter sequence for gBB4O is eukaryotic in origin. However, it will require the presence of additive viral factors for efficient expression. Therefore, the use of different promoters is desirable. The H8V-1 sequence of pKBXX contains restriction sites at two sites in the H8V-1 insert (e.g. Hindll and E
coRI) can be easily recombined on eukaryotic expression vectors. B,p! Expression from p52BXX To express gB from p52BXX, the plasmid is
31 (5' to 3', Either the 3' to 5' end is treated with an enzyme preparation containing an exonuclease active component.To remove the hydrophobic sequences (starting at approximately 3040) that bridge the membrane, the DNA is
is cleaved using XhoI. XhoI specific site present at +2672. The XhoI site at 1 is p528
In the XX construct, the 5alI-Xhol hybridization site was changed. ) Bal 31, XhoI fragment (plant-terminated) was identified by agarose electrophoresis and gel eluted into SmaI, 5alI cut pUC8D.
It was ligated with NA. In pUc8, the multiple cloning site, in other words, Eco RI - Sma I - Bam HI -
8a]I-pstl-HlndllI is pUC9
The direction is exactly opposite to that in (Table 3). Other points are exactly the same. The direction of β-gala, tosidase RNA and protein synthesis is from left to right. H8V-
The two sequence insertions are from Bal 31 to XhoI. The direction of composition is also from left to right. Of particular note is that Sma I leaves the plant end. Sm
At the a I-Bal 31 junction, all three decoding phases are expected because the Bal 3] exonuclease cleavage does not occur simultaneously. 5alI (GTCGAC) and H8V-2 in the multiple cloning site of pUC8
Because both CTCGAGI and CTCGAGI cut after the first nucleotide of the codon, the correct decoding phase is preserved at the 3' attachment point. Those codons that protect the β-galactosidase decoding phase at approximately
It will accurately express the gB peptide fused to galactosidase amino acids. Since the decoding phase at the 3' H3V-2/β-galactosidase interface is preserved, the gB sequence is now preserved as a result of being fused at amino acid residue IO. In essence, β
The four amino acid residues of galactosidase replace approximately 500 of gB. The fusion protein uses the endogenous β-galactosidase terminator present in the vector, which adds approximately 100 amino acid residues to the C-terminus of gB. The ligated DNA is used to transform E. coli (strain RDP211) and ampicillin resistant colonies are obtained. The size and orientation of the H8V-2 insert in these clones is determined by restriction mapping. The average insert size was found to be approximately 1.5 kb, and all inserts had the correct orientation. Beta-galactosidase cleaves lactose (a trisaccharide) into glucose and caractose, which provide energy for cell growth. Its synthesis is regulated at the stage of RNA synthesis, with or without lactose (or non-metabolized, chemically analogous unnecessary derivatives, such as isopropyl-β-D thiogalactoside fIPTG). The regulatory nucleotide sequence is the lac promoter

To identify clones with in-phase gB/β-galactosidase fusion products, cultures were performed in the presence of IPTG and phase microscopy. (Total culture ratio 400 times)
) was tested for the presence of Some foreign proteins (particularly hydrophobic proteins, produced in large quantities by E. coli) will be collected in easily detectable inclusions such as the armpits of bacteria. contained contained objects. In these clones, the mitotic or inhibited, cylindrically shaped cells become filamentous, with inclusions present in more or less regularly spaced spaces along the fibers. The culture is
When grown in the absence of inducers, it appeared normal. Samples from cultures grown in the presence and absence of IPTG are prepared for protein analysis using sodium dodecyl sulfate l5Ds)-polyacrylamide gel electrophoresis. The cells are pelleted by centrifugation and then lysed. The lysis mixture contains the supernatant containing cytoplasmic proteins [soluble] and
The precipitated supernatant proteins are then precipitated by acid treatment and pelleted to separate membrane proteins (insoluble) in the pellet. The pellets obtained from the cytosolic and membrane fractions are resuspended in equal volumes of electrophoresis sample solution and the fractions are subjected to SDS electrophoresis. The main binder was 5 in the membrane fraction of induced cultures.
A mobility consistent with 5 kd is observed. this is,
gB// is the expected size for a galactosidase fusion product. No conjugate is detected in the cytoplasmic fraction obtained from induced cultures or in the membrane or cytoplasmic fractions of uninduced cultures. Contains inclusions grown in the presence of IPTG. Membrane fractions from 17 independent clones were approximately 65
The main binder was given a mobility of kd. By use of standard marker proteins, it is determined that 15-50 μg fusion product protein is obtained from each me of culture containing the equivalent of approximately 5×10 8 cells. The immunoreactivity of the 65-kd fusion protein is tested by transferring the electrophoretically separated proteins onto a sheet of nitrocellulose, again using electrophoresis as the driving force. Electric fronting (elect
roblottingl is a tool for plotting (blot
ting l equipment as determined by the manufacturer. The nitrocellulose sheet containing the protein is soaked in a commercially available preparation of antibodies against IISV-2 or HSV-1. These antibody preparations contain the IgG fraction of serum obtained from rabbits. The antibody is shown to bind preferentially to the 5kd protein and detected by an enzymatic reaction. The second antibody, Tsuzuri Yaki anti-Tsaki IgG (the IgG
(loss of horseradish peroxidase co-unbound with)
will bind to the added and previously bound rabbit IgG. Finally, the substrate for the enzyme was added, causing the formation of a blue colored precipitate at the site of the enzymatic reaction. A precipitated blue conjugate is observed with a migration rate consistent with 65 kd in the nitrocellulose blot (blot 1). Its presence is only detected in the membrane fraction of cells grown in the presence of IPTG. Although the 65 kd protein does not react as strongly as the HSV-2 antibody, it does cross-react with the HSV-J antibody. One of the clones containing a plasmid called p52ΔNX-60 was selected for further study. Selected.HS
The 5' and 3' ends of the V-2 insert were recloned into the M13 cloning and MI3 sequencing hectors, and the DNA sequences were compared to the 5' and 3' junctions in p52ΔNX-60. The point was determined. H.S.V.
-2 insert from 135 to 6291 of HSV-1gB
contains sequences based on the corresponding amino acid residues (Table 2
]. This was performed using restriction endonuclease analysis (1,5 kb
65 kd l of the fusion peptide. This insert is shown to be in phase with codons characterized by β-caractositase at the 5' and 3' attachment points. For animal testing of the 65 kd peptide product from p52ΔNX-GO, large amounts of protein are required. The separation procedure for membrane fractionation is scaled up at a constant rate and m2 quantities of the 65 kd peptide are placed in the preparative electrophoresis apparatus. This device converts proteins from the bottom of the gel into PaNg
and electrode buffer
fractions can be collected. Fractions are fractionated by analytical SDS electrophoresis ()1/L, the peak fraction containing the fusion protein, Q-1, is pooled and dialyzed against the bouquet. A: Unshrinkable, eaves made of A11 6
5 kd protein intensity is 100 to 200 μg
/d. This concentration is suitable for introduction into animals after dilution 1:1 with Freund's adjuvant. The 65 kd protein extends in the 5' direction towards nucleotide 1192. (Table 2) It is also desirable to isolate DNA clones containing more nucleotide sequences in this xylem. For this, the smaller 13al 3
] A clone with the generated and deleted part is created. H.S.
Expression clones derived from pKBXX of [V-] can be created using similar procedures. 4 8 2 N
The ru1 site is common to HSV-1 and HSV-2. Example I H8V into pBR322 to make BamHI-G
l (KOS strain) Cloning of BamHI fragment A, restriction endonuclease treatment Eppendorf vial
l ) (made of plastic and has a snap cap)
pBR322 (Bethesda Research Institute) 1 μt (
approximately 0.5μ2), KO336μt (approximately 5μ2), Bam
HI3μt (8 units/μt) is 1 part restriction buffer 1 part
Digested in 10μ. Restriction buffer 1 has a pH of 7,
4 in 10 mM Tris buffer, 50 mM sodium chloride, 10 mM magnesium chloride, 10 μM mercaptoethanol, 100 μ'/mt nuclease-free bovine serum albumin. Digestion is carried out at 37°C for 3 hours. (It should be noted here that an excessive amount of amino acids was used and extra digestion time was allowed.)
must be understood to be disconnected. One unit of enzyme will completely cleave 1 μ2 E. coli lambda phage DNA in 1 hour at 37° C.). After digestion, the reaction mixture was dissolved in T E (1 m MFJDTA
, 400 μt using 10 mM Tris, pH 8, o)
diluted to The solution is heated at 65° C. for 15 minutes to inactivate the restriction endonucleases and then cooled to room temperature. The solution was then diluted with chloroform 24:3 to inactivate and remove any residual enzyme.
- 1 portion of methylbutanol is extracted twice with equal amounts. (Methods other than inactivation of restriction endonucleases by heating include
Sodium dodecyl sulfate (SDS) to a final concentration of 1%
It is to add. Both SDS and inactivated enzymes are separated from the DNA by passing through a small Sephadex column. After transferring 240 µt of the remaining solution to another plastic vial, add 60 µt of 5M sodium chloride and 500 µt of chilled ethanol (approx. amount) is added. The mixture is kept at -75°C for 30 minutes and centrifuged. The supernatant was then removed and the DNA pellet (p611st)
is vacuum dried to remove the ethanol. B, The dried pellet of the ligated ly+tM adenosine triphosphate 1
7 μt and 100 units of T4 DNA licase together with 20 μt of ligase buffer (50 m
M Tris buffer, 10mM magnesium chloride, 2
(0mM dithiothreitol, 50μ2/μt containing nuclease-free bovine serum albumin). This mixture is incubated for 15 hours and used to transform E. coli RR1. A 20 mt culture of C1-transformed E. coli was grown at an optimal density of 03 (620
nm ) in medium, saturated with carbon dioxide gas, and grown at 37°C. (V medium is tryptone 101'm, yeast extract 55'
m, NaCJ72m, C1,5ym, My So4
．． H2O0,59ff! , containing 1 t of water), the culture was chilled on ice for 30 min from lO, and the cells were centrifuged (containing 1 t of water).
Pelletize at 600 (M/X 5 min) at 4°C. The pellet was added to 20 ml of ST cooled to 4°C.
(25 m M Tris-Hα at pH 7.5, 10
Gently resuspend in mMNaCl). Cells were pelleted as above, 10 ml, cA
S T (25 mM Tris-Hα at pH 7,0, 10
mMNa C1+ 50 mMCaα2-2H20)
and kept on ice for 20 to 30 minutes. Cells were pelleted again and placed in a 1.35 m Eppendorf vial.
A. Very gently resuspended during chilled (4° C.) CAST and stored chilled until used for transformation (within 60 minutes from 0). Cell suspension in CAST 0.2 m7, CAST
plasmids (0,01 to 10 tt'i D
NA) 0.1 mA is added and the suspensions are mixed gently and kept on ice for 60 minutes. The mixture is warmed to 37° C. for 2 minutes and cooled at the same time interval. The contents of the vial were added to V medium 2.7 mA in a culture tube to give the plasmid DNA a chance to insert into the cells and for expression (the ampicillin resistance gene characterized by this plasmid). (from 2 to 3)
Cultured for hours under carbon dioxide gas saturation. At this time, the cells were diluted 5 times, 50 times, and 500 times with medium, and 0.2 ml of these aliquots was spread on the surface of V-amp vetribrate (ampicillin 25 my/
V medium containing L and enriched with agar 159 m/t). The plates are incubated overnight at 37°C and the colonies that grow are used to prepare plasmid DNA. Example 2 Plasmid pKBG-BS3 The Bam H knee G clone obtained in the previous example is subjected to restriction enzyme treatment according to the method of the previous example. In the representative reaction with 5atI shown below, the restriction buffer used was RB-8 containing: Tris buffer 6□M pH 7.4 Sodium chloride 150 mM Magnesium chloride 10th edition β-mercaptoethal 10 mM Thus, 10 μPBamHI-G is
Partially digested with approximately 1 unit of 5atI enzyme for 15 minutes at room temperature. The results in Example 2 were 0.387 SatI from 0.3458 amH1 site.
Coordinates to the site to produce fragment pKBG-BS3. Example 3 To obtain pKBXX, 10μ of pKBG=B
S-3 is completely digested with 8 units of BamHI for 2 hours, followed by partial digestion with 1 unit of XhoI in RB-1. In both cases, Restriction Buffer 1 is used. In a partial digestion such as that used here, 1 unit of enzyme is used for 10 μ7 of DNA and the reaction time is reduced to 15 minutes. In agarose electrophoresis, 0.345 to 0.37
4, O corresponding to the BamHI-Xh o I fragment of 2
A K b conjugate is obtained. This fragment is electroeluted from the gel, extracted using an equal volume of 24[deg.]l of a mixture of chloroform and 3-methylbutanol, and ethanol precipitated. This fragment is then ligated with BamHI-8aAI cut pUC9. Thus, pUC9 was first subjected to the action of BamHI and 5atI by the method described above, and then injected with H8V-1 at the corresponding site.
sequence (the electroeluted fragment above, o345BamH1 to 0.372 X h oI) and is ligated by the full force method of Example IB. Example 4 H8, V-2 (H
Cloning of EcoR1 fragment of G52 strain A, restriction endonuclease treatment Eppendorf vial
l ) (made of plastic and has a snap cap)
pBR325 [Becesta Research Institute] 2 μt (
04μ51), HG52 36μt (4μ9), Eco
R) 5 μt (40 units) are digested in 110 μ of Restriction Buffer 2. Restriction buffers 7 & 2 were 100 mM Tris buffer at pH 7.4, 50 nt M sodium chloride, 10mM
Magnesium chloride, 10mM mercaptoethanol 10
Contains 0 μy/mt nuclease-free bovine serum albumin. Digestion is carried out for 3 hours at 37°C. After digestion, the reaction mixture was treated with TE (1mM EDTA1p1
Diluted to 400 μt using 10 mM Tris (18 μm). The solution was heated to 65°C for 15 minutes to inactivate the restriction endonuclease, then
Cooled to room temperature. This solution is then extracted twice with equal volumes of a 24:1 chloroform to 3 methylphthanol mixture to inactivate and remove any remaining enzyme. After transferring 240 μt of the remaining solution to another plastic vial, 60 μt of 5M sodium chloride and 500 ttl of chilled ethanol (approximately twice the volume) are added thereto. The mixture is kept at −75° C. for 30 minutes, after which the supernatant is removed and the DNA pellet is sucked dry to remove the ethanol. B,') Ligated dry pellets for rho:/p52 KG-made tL- are resuspended in 25μ of licase buffer. (Licase buffer is
50 mM Tris buffer at pl+ 7.8. 20 μt of H8V-2 sample was prepared with 10 m M magnesium chloride, 20 mM dithiothreitol, 250 μ7/μt nuclease-free bovine serum albumin,
BH325DNA2μ410mM adenosine triphosphate 1
．． 5 μt, T4DA ligase (Becesta Research Institute Mortuary 2)
02) 50 units and mix. This mixture is incubated for 16 hours and used to transform E. coli RDP 145. Example 5 pUc9 for p52 E'G-B H&shading
Cloning of the amHI-Hindl [fragment from p52EG into p52EG2011A-(
Approximately 5μ2) was incubated with 2X Restriction Buffer 122 for 90 min at 37°C.
μ in J(amHI, Hin d, ut, Ec
. Digested using 1 μl of RI. Then i amount of 1M
Tris is added and incubation continues for an additional 90 minutes. In a second vial, 1 μt of pUC9 was added with 45 μl of restriction buffer] B a mHI , H3n
The cells are incubated at 37° C. for 3 hours using 1 μtf of dIll. Restriction buffer], (Re5tiction buffer
r 1 ) contains ]0 mM Tris at pl+7.4;
The buffer is otherwise similar to Restriction Buffer 2. After digestion, the cut DNA is separated by electrophoresis in a 0.75% agarose gel. The target conjugate is electroeluted from the agarose, then extracted as in Example 4, ligated, and used to transform E. coli. Example 6 Isolation of p52BXX from p52EG-BH In the first vial, p52gG-BHIOμt (approx.
) is restriction buffer 1 40 μ, /, +: IQ, B a
m J(], + 1 μl for Hind and incubated at 37°C for 2 hours and 30 minutes. Then,
A ho11 unit is added and the culture contains two Xh in many molecules of p52EG-BH. ■ Lasts for 15 minutes to cut only one of the sites. In a second vial, 10 μt of pUC9 is digested with 1 μt of f3amHI and 2 μt of 5ajI in restriction buffer RB-84011 for 2 hours and 30 minutes at 37°C. The restriction buffer used was RB-8 and contained: Tris buffer pH 7.4 Sodium chloride 150mM Magnesium chloride 10mM Nuclease-free serum albumin 100 μl/mL β-mercaptoethanol 10mM DNA was separated by agarose electrophoresis, electroeluted, extracted, ligated and used to transform E. coli RD P 145 as shown in the previous example. The electroeluted p52BG-BH fragment was 0.
From 345 BamHI site, 0.353 X h o
4,6K extending to 0.372XhoI through I site
b fragment. It should also be noted here that since the Xho site has the same sticky end in S'a L I, the HSV-20,372Xho1 site is cloned into the 5at1 site of pU09. That's true. Example 7 Expression of the 1B Fragment from pKBXX in Bacteria A, Generation of the rB Fragment
g) and 10 μt of nazI restriction endonuclease were added to an equal volume of 2X BatI buffer (restriction buffer RB − without Naα), and the DNA was
In an Eppendorf Snap Cap Vial, 3
Digested for 16 hours at 7°C. Two DNA fragments (1,48
After separation of Kb, 5.20 Kb), the required 1,48
Kb is electroeluted from the gel. The DNA is extracted twice with equal volumes of a 24:24:1 mixture of phenol, chloroform, and 3-methylbutanol, respectively, to inactivate and eliminate any remaining BatI enzyme. The aqueous layer containing the DNA was treated with sodium acetate (3M stock solution).
Precipitate by adding twice the volume of ethanol, then cool at -75°C for a 30 minute storage period. Gently pour in ethanol, suction dry the DNA, and incubate at 500 ml.
To 25 μt of bovine serum albumin of μy/μt, an equal amount of 2
40 m of X Ba L 31 buffer (pIIs, o
M Tris, 24 mMcaC1!2. 24mMM9
α2゜400mM NaCl, 2mM EDTA
) and 2 liters of Bat31 (2,5 units). These concentrations of enzyme and DNA result in approximately 100 nucleotides/min of hydrolysis from each end of the DNA. Bat31 preparations contain both 5' to 3' and 3' to 5'
Exonuclease degradation activity (
exonucleolytic activity)
therefore, both strands of DNA are hydrolyzed. 5 μl samples were removed at 20 second intervals during 3 minutes from O, and 50 μt of phenol-chloroform-3
Added to an Eppendorf snap cap vial containing methylbutanol. (See previous paragraph) Pooled DNA fragments are extracted, precipitated and resuspended in 50 μt TE as previously shown. Additionally, to remove residual mononucleotides with small variations in the sample volume, it is passed through a Fadex small column by rotary dialysis. To this, 1μ (approximately 0.1μ9) of pUC9 vector DNA, which had been plant-terminated by culturing with HinclI, was added.
is added. Typically, 5 μt of IOX ligase buffer [ ], see Section 4 (Example 4) for X ligase buffer] with 10 μM ATP is combined with 3 μt of T
4 DNA licase (4x10s units/mt). The resulting solution is incubated for 16 hours at room temperature and used immediately to transform E. coli as in Example IC. B. Antigenically active IB fragment Bacteria from unique colonies obtained from the above transformation experiments were used to be placed in 10 ml cultures and grown to an optical density (OD) of 0.05 . jT-[1111 from each medium was pelleted and 10
% trichloroacetic acid (TCAj) and the precipitate is washed in an Erapendrus snap cap vial using the same volume of 10% TCA. Finally, the pellet is washed in 00 μt TE. and p
H8,02M Tris-base
Neutralized by adding 2 μt. 10 μl of sample is applied to a nitrocellulose filter and air dried at 37° C. for 30 minutes. Bacterial proteins and DNA will remain bound to the filter. Nitrocellulose strips (145 x 190 mm) in 117.5 10 mM Tris buffer A (1). 17 (containing 1mM Naα, 0.1mM phenylmethylsulfonyl fluoride, plus 0.01% SDS and 10mM magnesium chloride) at 5 mA. The DNA is digested for 10 minutes by adding 0 μt of 2 ttq/mA deoxyribonuclease I (DNA5el) solution. All treatments of nitrocellulose are carried out in 8×150+mn screw-cap tubes. The nitrocellulose itself is wrapped inside the test tube. The strips are maintained by immersing them all in continuous culture medium by rotating the test tubes on a drum rotating at approximately 10 rpm. DNA se ■ and nucleotides are OD】%S
It is removed by washing twice for 5 minutes each with 5 ml of buffer A containing DS. Non-specific binding of H8V-1 antibodies is blocked by treating the nitrocellulose with buffer o, A 3 ml containing 0.01% SDS and 10% horse serum. The treatment is carried out for 3 hours. The strips are washed twice for 10 minutes each with buffer A5mt containing 001% SDS. H8V-1 antiserum (typically obtained on the basis of precise scientific principles) has been shown to contain fB in serum due to the presence of some antigenically active fIB fragments present in bacterial lysates. Added to bind antibodies. 10μ
t antiserum was added to 500 μt of solution B (1?MMEDTA
, 0.1% SDS, 0.1% Triton X-100, and 4% horse serum) and incubated at room temperature for 16
Time combined. After washing twice with 83 ml of solution for 10 minutes each, 2μ
Ci 1251 protein A [New England nucleus (
500 μl of lysed iB containing New England Nuclear) is added. Protein A is from Staphylococcus aureus and binds to a certain region of the ■2G antibody. Protein A is allowed to bind to these sites for 3 hours at room temperature. Finally, the filter
It was washed 5 times for 15 min each with 85 mA of horse serum-free solution, dried, and incubated for 24 to 72 h with X-OmatX.
exposed to line film. To test fB products synthesized in bacteria,
The 1B peptide synthesized from Bat31 purified DNA (Example 7) and the final clone preparation described below (see Example 8) were synthesized by radioimmuno-reaction of SDS electrodynamically released peptides. Can be tested. 1B
Because the product is under the control of the taC promoter, its production must be induced by chemicals that induce the synthesis of tac protein (such as IPTG). A 10 mt culture of bacteria was grown to 0D05 in the presence or absence of PTG, and the cells were pelleted (p
10% cooled TCA
1 ml and cooled for 30 minutes. 2
After the second centrifugation, the cells were washed with 10% TCA and the final pellet was washed at pH 8,0.
The electrophoresis sample solution (5mM Tris, pH 7.0,
2% SDS, 5% β-mercaptoethanol. Resuspend in 100μ of 0.005% Bromophenol Blue). A 10 μt sample is applied to a 9.5% gel and the peptides are separated at 100 volts for 6 hours. The peptide is electroblotted onto a nitrocellulose sheet and the sheet is air-dried. The 2B peptide is visualized by treating a nitrocellulose sheet as shown in Example 7 to assay for the presence of p,1i'B by radioimmunoassay. The predicted dog-like peptide should be visible. To prepare the yB fragment for use as antigen for rabbit breeding, induced bacterial cultures are treated with TCA and resuspended in electrophoresis sample solution as described in the first paragraph. However, the total volume is increased 10-fold to obtain approximately 100 μ2 of 2B peptide. fB from appropriate areas of the gel is mashed to a pulp in Freund's adjuvant and inoculated into rabbits for antibody production. 25μ2 of peptide can be applied once in the first and subsequent second injection. Antisera from rabbits are then tested for IB precipitation and virus neutralization ability, thereby confirming the desired fraction. Example 8 Expression of the 2B fragment from p 52 BXX in Hosogi A,1
Generation of B fraction Eppendorf snap cap vial
p52 in dorf 5nap cap vial
BXX DNA (25μ2m) was ethanol precipitated and
The pellet was washed with 70% ethanol, sucked dry and then washed with 45 μt RB6.
(100mM Naα, 6mM TrisH at pH 7.4
α, 6 mM MFα resuspended in 2.5mM 2-mercaptoethanol, 100μ'/mt-BSA). Restriction endonuclease Nrul (4 units/μ
4 μt of t) was added, and the mixture was heated at 37°C.
and incubated overnight in the presence of bovine serum albumin. A stop solution (5 μt) was added, and the reaction mixture was incubated with BioGel in TE buffer to remove inactivated enzyme, washes, and stains in the stop solution.
l) Subjected to two cycles of rotational dialysis using P-100. The DNA is ethanol precipitated, washed and dried. DNA is 25/It(DBSA(5
00 μy/mt) and an equal volume (7
) 2X B a l 31 buffer (pHs, o
) 40m M Tris, 24mMCaα2124 m
M M7C3゜400 mMNa Q! , 2-mM
3 μt of Ba/L31 (3,5 units) is added to EDTA). These enzyme concentrations and DNA9 degrees are D
It is responsible for hydrolyzing approximately 150 nucleotides/minim from both ends of NA. The Ba731 preparation contains both 5' to 3' and 3' to 5' exonuclease activity, so it can be used on either strand of DNA.
is also hydrolyzed. 7 μt samples each for 1 min 3
It is removed at 30 second intervals from 0 seconds to 4 minutes and 30 seconds.
Add to a vial containing 43 μt of E and 5 μt of stop solution. The extent of Ba131 cleavage is determined by running 10μ of the sample in an agarose gel (0.75% gel). DNA samples that appear to have been shortened by approximately IKb by digestion using Ba131 cleavage are subjected to two cycles of rotational dialysis. 0 and Xho of the final amount of l0XRB1
l (15 units /1i7) 1 μt is added,
The mixture is incubated for 8 hours at 37°C. Xhoi cuts DNA at nucleotide 2672, producing two fragments. 5 μt of stop solution is added and one portion of DNA from each time point is added to each of two adjacent blocks on a 0.75% agarose gel. Electrophoresis is run overnight at 54 volts. The DNA is visualized by ethidium bromide staining and the 1.7 Kb ligation is released from the gel and electroeluted (3 hours at 108 volts in 0.IX electrode buffer). Ethidium bromide was mixed with an equal volume of n-butanol (approximately 3 ml) saturated with TE.
) is removed by extraction three times. Extractions are performed twice with equal volumes of chloroform:3-methylbutanol (24:1), and the DNA is ethanol precipitated, washed with ethanol, and dried. The DNA was resuspended in TE50μ and 4μ9 p
trcs vector DNA (in advance, Wang mai and 5ail
(exposed to continuous digestion with) and subjected to two cycles of rotational dialysis. Vector DNAs were ethanol precipitated, washed, and washed with 48 μt Smal buffer (Tris, Hα (pH 8,0), 6 mM Kα; 20 m M:M α2,2 mercaptoethanol. 6 mM; BSA, 100 μM/ml). The sample is vacuum dried prior to resuspension. 2 μt S m
After digestion, the DNA was subjected to two cycles of rotary dialysis [in some cases 'Sm a l was chloroform-3 prior to ethanol precipitation and drying of the DNA. It is inactivated and removed by methylbutanol (24:1) extraction. ] In both cases, the DNA has an RBS of 46 μt.
411t S a L I (]0uni
ts/μm) is added and incubation is carried out for 2 hours at 37°C. H8V-2 DNA fragments obtained from each time point (
Approximately 2tty) is 0.2,1(7)plJC8,,D
Can be mixed with NA. 1 volume of 10X licase buffer, 1
A T P of μt (11ME//mt). and 3 μA of T4 DNA ligase (4×1o5uni
ts/ml) was added, and the total reaction volume was 50
Ligations resulting in μt are performed overnight at room temperature. The reaction mixture was prepared using the technique of Example 1c.
It is used immediately to transform 11. Identification and Immunological Activity of fB-β Galactosidase Fusion Proteins Individual')) Bacterial colonies were isolated from plasmids (of the class called p52ΔNX) with appropriate H8V-2 inserts.
and by inserting a containing proboscis that grows in the presence of l PTG (isopropyl-β-D-thiocalactocyto). Cultures (10 ml) from these colonies were approximately 1 O
D, induced state (56 mf/in 50% ethanol)
ml of 1. P T G 4 μt) or uninduced state (20% (V/V) glucose bath 150 μt)
In, ■ it grows in medium. Cells were pelleted (60007 x 5 min),] Omy/m
Lysis buffer containing t lysozyme, 4 μ9/mt pancreatic deoxyribonuclease (DNasel) (
5mM Glucose, 10mM EDTA, pH 8
, 25 m of 0,0 M Tris-H (100 μl). After 30 min incubation on ice, the reaction mixture was vortexed, the ice was thawed by warming to 2°C, and 5 mt of TE was added. is added and the mixture is vortexed. The sample is precipitated to pellet membrane protein-heavy or insoluble proteins, leaving cytoplasmic proteins or soluble proteins (liposome-containing) in solution. be done (6
000rX10 minutes). Equal volume of chilled trichloroacetic acid (10
% W/V) is added to the supernatant for 30 min on ice to precipitate cytoplasmic proteins. These proteins were precipitated (6000iii'
0 min), electrophoresis sample solution (pil 7.0 min), electrophoresis sample solution (pil 7.
．． Resuspend in 100μ of 05M Tris-Hα, 2% SDS, 5% β-mercaptoethanol, 5% flomophenol flu, 5M urea). 5 to 10 μt of 2M Tris-base is added to remove any remaining trichloroacetic acid in the pellet. Membrane proteins present in the initial pellet are still solubilized into the 100μ of electrophoresis sample solution. A 12% acrylamide gel (analytical gel) was prepared by combining acrylamide (12ym) and N,N'-diallyltartaric acid in 100 ml of gel buffer (0.375 M Tris.HCl, 0.1% 5DS at pl+8.8). It is made by melting diamide (0.56℃m). Ammonium persulfate solution at 0.6 mA (water 9
17m for mA) and 30μt N, N, N
'N'-Tetramethylethylenediamine is added to 90ml to catalyze the polymerization process. The solution is degassed under suction and 4E, mt 2 is used to form each separating gel. Two glass plates (+ 5.9 X i 9 cm) are spaced apart by 1.2 mm spacers on each side and by a node 3 spacer at the bottom of the plates, , the solution is poured. The complete assembly is simultaneously held in a plastic gel stand held by clamps and the greens are sealed with acarose (1.62 mt to 100 mt of water). plastic blank
(Width 1Bcm, length';!,, 5cm + thickness 1
, 2 in) is inserted to create a smooth surface on the top surface of the gel. After the gel has been polymerized ( ] time),
The blank was removed and 15 mL of deposited gel (5%
Only contain acrylamide, pil 7.00012M
Tris, Hα, 0.1% SDS (used in the same method) is poured onto the top surface of the separating gel. A comb with 10 teeth (each 85 rams wide x 15.2 rams long) is applied to the deposited gel. After polymerization of the deposited gel (1 hour), the protein sample (5 μL to 10 μt) of electrophoresis 1 sample solution A was
Added to each hole made by the comb's teeth. Gels were prepared using Tris-glycine electrode buffer (0025M
Tris. Electrophoresis is performed in 0.192M glycine, 01% SDS, pH 8.5) for 4 hours and 30 minutes at 108 volts. After electrophoresis, the gel plates are separated and the proteins in the gel, which are stained, are collected. To visualize proteins, Coomarsse BrilliantBl was used.
ue ) Staining solution (0,25% staining solution 1 (1,2+X
Glacial acetic acid, 45.4 x yt/L) was added to the gel for 30 minutes, then methanol-vinegar water (5-7
, 5-87, 5) overnight. Proteins used in antibody reactions are unstained and 12
Separation was performed using electrophoresis on polyacrylamide-8DS gels and then blotted onto nitrocellulose paper. An electric suction device is used and suction is performed according to the instructions provided by the manufacturer. The collection is
Electrode buffer containing 20X (V/V) methanol (192
mM glycine, 25mM Tris-HC/! , IXS
D S , pH 8, 3KA) for 1 hour and 30 minutes at 6 o volts. After the protein is transferred to nitrocellulose, it is cut into matching strips into 100 mm diameter plastic Petri dishes for antibody binding measurements. The strip is 5
mA phosphate buffer ratio of saline solution < pss) (
x, 149mCaCl2.2H20,0,2FmKα,
1.02 FmNa HP 04+ 1.029mK
H2P 04.0.29tnKH2P o,, + 0.
19mM C12,6H20, 89mNaα, pif
7.4), with a washing time of 5 minutes each.
Washed off twice. For non-specific tying, use horse serum 5 hours for 1.5 hours.
This can be minimized by treating the filter strips with 5 mL of PBS containing 00 μt. 5mA
After washing twice with PBS, the strips were soaked with 100 μt of horse serum.
commercially available anti-H8V-
2 (20 μt) or anti-H3V-1 (50 μl) rabbit serum overnight. The antiserum was removed by washing three times with 35 mA of PB and the g22 anti was washed with 85 ml of PB and 10 ml of horse serum.
Added in 0 μL. The second antibody C is goat anti-rabbit lrG containing horseradish peroxidase. The second antibody will bind to the I'PG present in the first antibody (at a site on the H8V-2 protein). The nitrocellulose strip is exposed to the second antibody for 3 hours. After being washed 5 times with PBS, 5 ml of substrate solution (1 ml of 4-chloro-1-naphthol, lOmp)
of ethanol, 10 μt of 30% H20, 1,00 mA
water) is added until the color develops at the site of the indicator enzyme (horseradish peroxidase). All cultures are performed on a rotary shaker at 75 RPM. For the C07B fragment animal assay, the membrane fraction described above is prepared from one liter of the induced culture in order to obtain a large amount of 65 KbfB-β-galactosidase fusion protein. A clone harboring a plasmid called P52ΔNX-60 is used. The membrane fraction harboring the 65 Kb fusion protein was pipetted to 10 ml (from an equal volume of IL culture) onto a 10% polyacrylamide-8DS preparation gel. Electrophoresed (150 volts). The proteins are removed from the gel and fractionated (2=
2) are collected over a period of 6 to 8 hours. Fractions containing the fusion protein are identified by electrophoresis on a polyacrylamide-8DS gel of fractions from the prepared gel. 5
The 0μ sample migrates over the analytical gel and its migration is compared to that of the unfractionated sample. Proteins are stained and destained and visualized as described above. A relatively pure 65Kb fusion protein is obtained. Proteins are dialyzed against PBS and diluted 1:1 with Freund's adjuvant. The 65Kb protein is then approximately loOμ2/mtt
v#W. 1 ml of alcost is used to inoculate multiple sites on the buttocks and back of goats and rabbits for antibody production. The method used was 7
Five inoculations are given at weekly intervals with serum collection starting a week later. To demonstrate the protective effect in mice, 10 μt to 100 μt are inoculated into the same site in mice. After different time interval inoculations, lethal doses of HSV
-2 (5 x 105 plaque forming units (pfu)) are inoculated into the matching sites of mice. Moisture-protected mice show a remarkable increase in survival over controls. Table 1 H8V-1rH The nucleotide sequence and the surrounding region H8V-1 (KO8) and map units between 0.372 and 0.345 are shown.
Starting with 0.372Xh o 1 site, ending with 0345 map index BamHI site. The array is 5' to 3'
It is written up to. Estimated CCAAT''(,,,,
) and "TATA" (-) signal sites are underlined. The three possible mRNA start sites are:
Indicated by (). The first possible AUG translation initiation codon is indicated using a box and arrow. The end point of translation from the AUG codon is indicated by the letters END. Putative polyadenylation signal AATAAA (black square dot)
is shown, and immediately after the estimated point of polyA addition, (△)
The mark will be shown. Clarification of the specification = (no changes made) Table 2 Comparison of amino acid sequences between H8V-148 and H8V-2rB (7) What is written in the bottom column is the amino acid sequence of ISV-1fB. . The column above represents the nucleotide sequence matching H8V-19B. The row just above it is
Nucleotides compatible with H8V-2rB are shown; only those portions that differ from the missing nucleotides of H8V-1 are shown. The X indicates the undetermined nucleotide sequence of H8V-2. (H8Vt14B residues), 7-
The fourth column from the bottom shows the corresponding amino acid sequence of H8V-2fi'H at positions that differ from the nucleotides of H8V-11B. The top number is H8V-1
It is the number of amino acid residues of FB. It may be noted that five extra amino acid residues are present for H8V-2rB in the gaps shown in the H8V-1 sequence. Mouth ku>e1ML+-Ro0υ-RoLl <Oro u Kuro Ll>mouth eJ MLI -# Ml-m 15-
Low +15 LJ-Ro 1-++ Nl! + -mu
tz W6> aQ+, pm ua -I Ll-+
ww -X Kuko XΦh Mari LITh -<6 LJ
m low M I-at 〆Φ--<low-Oυ-<,
−+, J J:X←−xljv+ −〇− Hat rear
Ll! (, +g tJ e M +-661J←5
m LIl-m @l+l, LBu6M l-II
I>l LJ k -Ll l-U m 161J!
+ LJ < + Ll +MLI -XLJa a
LIl m dTh m dbl Llaxcl+
Ill M u + M l−Ll m ta < 6
LJ M 'Ll eM IJ , -X IJ j
l-1j ul + LI L Ell + cJ k
M ljm X -c +++lu ha m LJ
W Ll m Ll a-Φz X Lj > B
LJm) ωα −○ko ■ku. ml-451-, %lj+ l-Ll −+u+<x
III ←al Lll-1-IJ l-++X lj
W eL IJ u m M LIl < m >
u LJ , -u aa b< + X LJ 6 1
J s I-Ll < M Ll w LI l111
←l-m X LI +,, LJ + 4116 <
Th Ll j ha ＞-L+口Th XLJa
Low ψ ＜−＜− ←υ−υ←m XLJa −1j
++ -Ll k ++ ←-One bite! ＋×←ψ
Cap Φ -U to C-ji 〉) u LI 11 X
←t-6ta LJ +,, W tq < 11
Llw U-+ Ell ←j M! ++ 1. ,
LI LI U L, IJ < h <-< )
輔口←α X＜η asu←φ −Q〈−RI２ ←−１
ro1 nro 〇 −υω kuψ Uh ←υ−←Φ−X
IJ k −←+e−ih J− ←ni〉Φ←−bau
a l-++ LIl 〈-Q-←＋ Ll ljw
x←m 6 Ull e(Ija LJum euu
e−１Φ− MLJ−+Ulj> , −(ψ Q−−B−N+Rho III Φ−alJ←0 Graduation phrase Low −Qku−0Htll tJ−Ro 1T and ++ O−u
Ro Ro αL + tpffl Oum Ro tr<m Ro V
←Φshi 0 XU−0v υ−to+n<> ■ υ−−
-Ll-on lj-φ←LJa +1<-1 clause
−〇−−d roh ×roto ri〈Φψ U−←a
-LJ LIl M LJU Φ--＜A ← To the bow φ
Th u++ QLJ,-! L-Lll-LJLl,
+ -LjjL) ←-MljIILJm m+51J
eL ＜+4t5(+0←Φ-xO-kukokokuφ
Low Qα+ I-LI U M I-zu Pei -
Kushima tr+-<1-1←ψ Mlj>lj--ri<
−w m ＜勺ω←ww x←aI Φ−LOW kuφ
E + I-Ll ++ M Ll , -1j ++
LJ W < d La +6h LJm Mu, -L
Jh > Llam Ija -IJul'll+
tj ul −X←+I low −9kuψ −Ll +
+ V -←U-Ran us m X 15 > c -
-Ro - -■ Tsuyoshi ) ■ -> 1jLI Th
xt5) PeQ e<fj> a IJ> 411
φ藺 wba to XΦ−υ −〇Φ−−Φ− く−(
ue Xt5! LIW IALlt! ＋um Lu
ll-mLl ha M LI-1 phrase 2Uku- ■-
Ku-I+Φku- PaLI4 lj-ψkuri-Uw+
<XLl+MLl+=+Mtll+1ML1mLIW
U-Li eOL+-〇 Romiro←LlalRo Roro ■Roku ψ ψ Low (To) Ku-ro ←-~ υ4 Li Llj Ku--Lee--+ Ll-~ Ku- To Ku-to <-Ll e tll m' Ll k cj ψ
Ll w , Ll lj m < -(Th LI U
< ) LJ! Ll -υ-<-t1) <-<
+H++ m lj←＞ Ll ω ca+−LJm C co − Q phrase ■−−2 +2 >< M <−hat←υ−LIha
, l-α t++ low low >wmta5 (tl
l+11+, pw Lllj j all! tnΦw
a<-<> b+ ←-U l-41Th <■-(
-un, <-day (LJ, -+ <hat Uυ0 low Q
Cut Φ-Uζ ΦU-IJ t-< h <-←■ Kuψ U-ucL ←-Ll j ++ >< m d
mLl! M ta Q IJ > u! L.J.
e u Q■t−QL, ta + < −+FIl
lLI L. Ll +IIJ CL lj W lj ha ++
ml LI W! u+1. J m Ll 41
1j W LI LI LI m! <MuB Low ←
j < -tj + <>ku + w ← α low
LJ m < -1l16 Ll al 16 a L
l 11 u -Ll! 5L+ +-k-'II
To= ←−←QLJ a, < −To ←False 1
-5shi Q-Lull Lla C-υRo LIl Lokta++ +tφ To-υ-kuh Ku-←u+tm
+1! ＞LJm ←ν Low U-riff IJI+
←U+ ψ U-ji- (-(+ I-m <>
←ωU Tsuyoshi livlLh-■)<' -riliriichiro 1
jllllro 9-ro Uroro 0 phrase υaO-no -
t > IJ Ml Φ Llw +1 + -e'
l <00 phrase -<-onetoω -Uα N lj-bad
Φ〜ljo LJU Ij TO-LJ m w L,
Ll wU-←j I-m Lj + LJ, e L
l! To U To +- ca> ljm <-<-@
Ll -Ll +,, LJ be LJ-Kukoku-
←Cap Ll a ++ k LJ ++ <-U-cut> (++ LJ-←ψ H5-! RIll -1j
LJ-LJ a Ll! −〇ψ＜−1−１ ←−
<II Low <- H5be LI Th tll Th Ell m C
J m LJ j←LJ U ta m LJ cL,
+x 5 LI l-11u CL←j LJ -(I
I-e-ux Peψ←a Rishi Riichi 〇-←Q Same phrase QCtJ at < M Ll l-at lj -L
l aku- ←+ (for ←; ←-+tψ υ-<, -c-toα ■shi t5- ta -Ll > Riichi < Ll al Φ for u-
←+I ji-<to+low lj-mouth> LOM
k + < -++ ++ L) mC-Ljri Ql
−ta > Φφ υψ←−<−4> LIl −<
＞ −−Lj＞Ij−←−cut−＜−Ll l caw ljw 'lj+' +15− υ−←CL
JJ! LI J+ l-al ト41 Lj-←=
(+ '< +4 < s (g LIl−←1LJ
++ Ll + Ll +-1-++ u e ce
mLl −(Th IJ j l−4(ψ U−IJ
■ ←−＜−←False ＜−Φ−! ＋Lllllld! Ill +jll LJ←fu Qψmouthtp＜+p 0 w＜u 0 LJa 5 u
um 0 Ll-ro Q mouth■mouth, Llαjitsuυ-Ll■
ko++uLjriω LJ−ψ U←1 ω C1■ Φ
−+e LJeL ψ U−ψcI'IU ri + BeI
ll 0 0- ←-to kuψ ■ Φ--1 ←
φ ``Si O+h ri ←-ri low invitation
Q-■ω LIζ +6 @-me Qψ (company) U-
φ υ−bacteria−υQω kuψ o UJ++ N lj>
Size Q-■ U-ω-heap <-1/1<+
←U Re-low's C-Old> Robey-low 1jLl
+-1j + Ll e υ-Roro LJ IJ &
1 i-U (1Ilca a+ d V+ILB ←
Octopus
■ ＠RIA ΦRI 萄 ψRI ψ LIU＜＋ Kō
C4l −'s ←season toe Φ ←ω −←−1j
-15LJ- lj -C1l Φ-Φ Ll-1-
<-m LIU groan υh groan U-萄珈 low φko←U峨 shu. a own ←t −/1 lj+ t
υ＋ ■＠l ←υ −υU〈− ■ （−一ト ←へ ?−ljm ?−La can To υku− ψ−υku cheer
Phrase ψ−(u # to kl−←Uri Q−.φ ←
…←JIlJ+−←・ ←−＜h ←1 1− & IJ M Ll + u Th <−←al
ll m LI1- ta m Ll t-LJ!
Th 111Ll+ ←−” −(Th −Ill −
urLll ++ Lll b ψ−←−υ−−91I
I cJh ++EuaI ljh υ←−＜ ! W
Th Ll−ψ−hat ←+ ψ−ta k (−−−
riU←w w -ul < +ta w ta #L
ll m ha ulta gl Ll (Ll 1-
gl tJ l-U cs m Th ta1- a
La-1-! Ill:< ) −u+υ−1Jl
−←False To False Ku− −9 Lll Lt Th La + Lla LJ m L
I-! ca Ll (LΦ−←a LI k u +
Ll! -Iφljm <II LJa Ll++
+l +e+ M ljmkiri-tIllll υjin. V
υ−−0υ−Q−←+r −t v+ l−x t,
r z −Ll ta −La1 @ ljh < 1m
l-&< + -low uu iu + Ll &
u # L) (!LI Th-yo ←+@LI
U l-! i-at ta-←el-L5 glue ←shun ←to. -La-←al 4- ←u Ll -LI
L, < -dll < -+c-w La+l L
la cawLl m Ll h ←Ll u IJ
++ l−Ll w umυ−Uoka ←j Ll +
+e h < -ul Tl ta w ←ch Ll
m 1-+ ta w611 Ll -ω LI轡
匍 υ－ 小－Uc－ψ LIThVNLl
-W 1m us Llal ψm←-RO ←Q~
u-! ? -Inn? -+zTh +'-←ie tob
IJIII ao Ll -gl) ww eLI
a IJ hat w y w a LJ #tIllll(
++ co @ ←+11 LJ −Ill cheer ψ−L
l m LI m +, s + LI Th u X
u ThLIL lj−←−low kuhu u− 1Il−υ# ljh lJIM +t+ low LIW
LIIsh LITh LILl ul h LIL
. (Ill LLl-1j -Ll -1-11-La
LI ll1Jl hat m w tIlw cm ++ <
m w ta←-un tIlw 1j-LILa
L+low umu+, l-U +Ja+ I-U u
t-4l-Ll -M um tJa LllJ+ L
I-1-Llal h Llam Iwx lj~ w
-Qn <++ w tanLlal tau) Ll
m wz M ul-Th LIIO←-tll -L
l -1-11+-tIllll -ulLlu<-ul
M IJ−υt+ −υ< m M ta LI I
LLl−LIl+ tl++,, ul a <ta
c a Ll g←-1-U Ll at 1-+a+
< -w kl < nts Llal h1j E ta tohc++ 5 False Ll 1-OA Example ←
-Q-←lI φ鴫LJlLl a IJI勺all
-c++ +! ＜ u−υQ(−ku ←−t−4
Table 3 Multiple cloning sites in pUC9 and pUC8 Multiple cloning sites enriched with the indicated restriction sites are indicated in the figure. The cloning site is closely related to the N-terminus of the β-caractosidase gene, with 11 amino acids located between amino acids 4 and 5 of the protein compatible with pUC9 and between amino acids 6 and 7 compatible with pUC8. This will cause it to be inserted. The new amino acids are derived from the normal β-galactosidase amino acids. RNA
and the direction of protein synthesis is from left to right. 12345 1 2 3 ' 4 PROSERL[ill ALA
^LATIRMET ILE TIIR ATG ACCATG ATT ACG CCA AG
CTTG GCT GCA' Hand III'
Psl1 123456123 THRMET ILε TIIRASN SERa+4
gIy 5erATG ACCATG ATT AC
G AAT TCG CGG GGA TCCEco
R1 [lam II ea 1 ss 1 Note: TTC is amino v6-serine 67891 above the arrow
011 GLY ARG ARG )LE PROGLY 5
6 7 8ASN SURLEU ALA GGT OGA CGG ATCCCCCGGG AAT
TCA CTG GCC'Bosll' EC
0RI 'SaL I Sso+ LCCI X+eo! Hind II 4 5 6 7' 8 9 10 11 7 Itva
t asp Ieu gln pro ser Ieu
ala LED ALA. GTCGAC'CTG CAG CCA AGCTTG
GCA CTG GCCPsi l 1lind I
II Encrypt. Procedural amendment (method) % formula % l Incident indication 1982 Patent application No. 129915 3,
Relationship with the case of the person making the amendment Patent Applicant Name: Stanley Person (4th name, agent) (11) We will submit one copy of the specification (pages 78 and 79) as shown in Attachment 11.

Claims (1)

[Claims] 1, 1 (antigenic for H8V-1 or H8V-2, comprising a sequence corresponding to the amino acid chain found in glycoprotein B of the SV-1 or H8v-2 virus) A non-glycosylated amino acid chain. 2. The amino acid chain of claim 1, wherein the virus is H3V-2. 3. The amino acid chain of claim 1, wherein the virus is HSV-1. 4.750 5. The amino acid chain of claim 2 comprising no more amino acid residues. Amino acid chain of range 4. Not less than 65 amino acid residues J, (: /, including!1
) the amino acid chain of claim 4, containing amino acid residues 135-629 as referred to in Table 2, and having a molecular weight of about 65,000 daJ tons; Amino acid chain. 8. The amino acid chain of claim 3 having no more than 750 amino acid residues. 9. The amino acid chain of claim 8, comprising nine sites: an N-terminal hydrophobic leader, a membrane bridging sequence, a C-terminal ionic sequence, and a glycosylation reservoir. 9. The amino acid chain of claim 8 comprising not less than 105 amino acid residues. 11. The amino acid chain of claim 10 containing amino acid residues 165-629 as referred to in Table 2 and having a molecular weight of about 65,000 daJL tons. 12. ) contains a sequence corresponding to the amino acid chain found in glycoprotein B of the ISV-1 or ) [5V-2 virus and is antigenic in response to 1 (SV-1 or H8V-2). , a lid to carry a DNA containing deoxynucleotides encoding a non-glycosylated amino acid chain.
0 13, containing a sequence corresponding to an amino acid chain found in H8V-1 or H8V-2 viral noglycoprotein B, and containing a non-glycosylated amino acid chain that is antigenic to H8V-1 or H8V-2. A portion of DNA that consists essentially of a coding DNA sequence.