JPH0198486A - Cloned prepro-human serum albumin gene and human serum albumin gene, plasmid containing these genes,microorganism transformed by plasmid,and production of prepro-human serum albumin by using said microorganism - Google Patents

Abstract

PURPOSE: To produce a large amount of prepro-human serum albumin by the use of microorganisms transformed plasmids containing human serum albumin genes.

CONSTITUTION: This cloned prepro-human serum albumin gene is obtained by grinding human liver cells in the presence of a ribonuclease-inactivating substance, removing solid cell pieces by a centrifugal separation method, etc., precipitating RNA from the obtained supernatant, subjecting the precipitated RNA to an affinity chromatography using oligo dT cellulose to separate human serum albumin mRNA from non-adenylated RNA, synthesizing a cDNA by the use of the mRNA as a template, inserting the cDNA into proper cloning vectors (preferably plasmid pBR 322), transforming microorganism cells with the cloning vectors, screening the obtained clones with e.g. a rat serum albumin cDNA, and subsequently culturing a positive clone in a proper culture medium.

COPYRIGHT: (C)1989,JPO

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a cloned preproduced human serum albumin gene, a plasmid containing these genes, a microorganism transformed with the plasmid, and a microorganism transformed with the plasmid. The present invention relates to a method for producing prepro-human serum albumin.

BACKGROUND OF THE INVENTION Human serum albumin (sometimes referred to below as ISA)
is the main protein component of plasma. This protein is produced in the liver and is primarily responsible for maintaining normal osmotic pressure in the bloodstream. It can also bind and transport various small molecules through the blood.

ISAs are administered in a variety of clinical situations. For example, shock and burn patients typically require frequent administration of ISA to restore blood volume and thereby improve some of the symptoms associated with the trauma. Patients with hypoproteinemia or erythroblastosis fetalis may also require treatment with serum albumin.

Today, ISA is primarily produced as a by-product from fractionation of collected blood. The disadvantage of this is that cost and blood supply can vary widely. Blood may also contain undesirable substances such as hepatitis viruses. Therefore, it would be beneficial to develop alternative raw materials for ISA.

Winter-type behavior is known in human serum albumin.

Protein electrophoresis reveals over 20 ISA genetic variants (Weitkamp et al., Ann, Hum, Genet, London).
on) 36+381-391 (1973)). The amino acid sequences of these variants have not yet been compared, and their distribution in humans is unknown.

(Problems to be Solved by the Invention) Therefore, an object of the present invention is to produce human serum albumin in microorganisms. A further object of the invention is to produce ISA economically. Another object of the present invention is to develop a cloning method that can be applied to other serum proteins.

Means for Solving the Problems According to the present invention, a novel human serum albumin gene has been cloned and microbial expression of the gene has been described. The nucleotide sequence of the full-length H3A gene and the amino acid sequence of the polypeptide specified by the gene are also reported in the text.

The procedure for providing ISA-producing microorganisms can be divided into the following steps, which are fully disclosed in the text. That is, (1) collection and isolation of H3A mRNA, (2) using mRNA as a template and complementary DNA (
cDNA) in vitro; (3) insertion of the cDNA into a suitable cloning vector and transformation of microbial cells with the cloning vector; (4)
) Recovery and isolation of cloned genes.

Full-length cloned H3AcDNA using the method disclosed in the text.
A can be isolated.

Nucleated cell genes are contained in the chromosomal DNA of the cell nucleus.

This chromosomal DNA exists in a small nuclear protein complex called chromatin. Nucleated cell chromosomal DNA contains coding sequences (exons)
Intervening sequences [intron (1n-tron)]
including. It is not expressed correctly in cells. To this end, a preferred method of producing contiguous coding blocks for a particular protein requires the use of transfer RNA (mRNA). The transfer RNA has a ribonucleotide sequence corresponding to the gene of interest without introns and can be conveniently recovered from nucleated cells that produce the protein specified by the gene.

Human serum albumin mRNA can be recovered from human hepatocytes in usable amounts. H3A mRNA produced by hepatocytes is complementary to one of the double strands of the H3A gene, and as described later in the text, complementary DNA
(cDNA) can be used as a template for synthesis. cD
To effectively use mRNA for the synthesis of NA, it is conveniently recovered from cells in a relatively pure form. McCandl 1ss et al., Methods in
Enzymology (Methods in Enzym)
The guanidine thiocyanate/guanidine hydrochloride extraction method described by U.S. Pat. RNA is inherently less stable than DNA and is particularly subject to degradation by bonucleases present in cells. Therefore, mRNA recovery methods generally employ means to quickly inactivate any bonucleases present.

Generally, recovery of total RNA begins by disrupting cells in the presence of a bonuclease inactivating agent. Cell disruption can be carried out by contacting the cells with a lysing agent, by freezing/thawing, or by mechanical disruption, preferably by a combination thereof.

A mixture of guanidine thiocyanate and a reducing agent such as mercaptoethanol has been found to function effectively as a ribonuclease inactivator (McCandliss et al., supra).

After cell disruption, solid cell debris is removed, for example, by centrifugation. RNA is then precipitated from the resulting clear liquid. Precipitation is accomplished by known techniques, for example, by adding a water-alcohol mixture (e.g., ethanol) to the solution in an amount sufficient to cause precipitation. The RNA is then resuspended in guanidine hydrochloride solution and precipitated with ethanol twice in succession. At this point, the RNA is of high quality and does not contain protein or DNA.

The next step is the separation of mRNA from total precipitated RNA. Human serum albumin mRNA is polyadenylated,
Therefore, oligodeoxythymidylate (,tlJgodT
) can be easily separated from non-adenylated RNA by affinity chromatography using cellulose (Aviv, et al., Proc. Ac
ad, Sci, USA) 69+1408 (1972
) ; McCanditss et al., supra). Total RNA is applied to the column in a solution containing approximately 0.5 M sodium chloride. Only under these conditions, polyA'' RNA binds to oligo-dT cellulose and can be specifically eluted by washing the column with salt-free solutions.

To increase the preparation for HSA mRNA, BoIJA
``RNA can be size fractionated by sucrose density gradient centrifugation. RNA activity in the various density gradient fractions was determined by in vitro translation in reticulocyte lysate (lysaLe) (Pelham, H.).

H,) et al., European Journal of Biochemistry (Eur, J, Btochem, )
67, 247 (1976) or by electrophoretic analysis of protein products (Laemmli, Nie.
, U, ), Nature 227.68
0 (1970)).

Once a protein of the size of H3A can be synthesized, IJA''
If RNA can be isolated, it can provide a template for cDNA synthesis. This technique involves enzymatically constructing double-stranded DNA having a nucleobase pair sequence identical to the coding sequence of the original chromosomal gene.

cDNA does not contain non-informative segments (introns) within the coding region that may be present in nucleated cell genes. It is thus ultimately transcribed and translated in prokaryotic systems.

The synthesis of H3Ac cDNA uses reverse transcriptase, the Klinow fragment of DNA polymerase 2, and the enzyme S1 nuclease (Kacian, De.
ian, D., et al., Proc. Nat' 1. Acad.

Sci, USA) 73.21901976) McCandliss, R., et al., Methods in Enzymology.
in Enzymology) 79.601 (198
1)). Reverse transcriptase catalyzes the synthesis of single strands of DNA from deoxynucleoside triphosphates on an mRNA template.

The polyr(A) tail of mRNA is approximately 1
An oligo (dT) of 2 to 18 nucleotides is a cDNA.
Allowed to be used as a primer for A synthesis. The use of radioactively labeled deoxynucleoside triphosphates facilitates monitoring of synthetic reactions. In general, α3Zp-containing deoxynucleoside triphosphates can be advantageously used for this purpose. cDNA synthesis generally involves the synthesis of mRNA, deoxynucleoside triphosphates, and oligo(
dT) and reverse transcriptase in combination. The solution is heated at an elevated temperature, e.g. 40-50"C, for a sufficient period of time to effect cDNA replication, e.g. about 5-50"C.
Incubate for 20 minutes. The reaction conditions are basically those of Kacian, D.L.
(mentioned above). After incubation, ethylenediaminetetraacetic acid disodium salt (hereinafter Naz.EDTA) is added to the solution, and the solution is extracted with phenol:chloroform (1:1 by volume).

The aqueous phase is advantageously purified by gel filtration chromatography,
The cDNA-mRNA complex in the eluate is precipitated with alcohol.

mRNA was extracted in the presence of cDNA with dilute sodium hydroxide (
about 0.1 M), high temperature (e.g. about 60 to 80°C), about 15
Specifically hydrolyzed for ~30 minutes. Neutralization with alkaline solution and alcohol precipitation yields -end strand cDNA copies.

- End-strand cDNA copies were shown to have a 5'-poly(dT) tail and a 3'-terminal hairpin structure resulting in a small piece of overlapping DNA (Efstratiadis, E.
(Efstratiadis, A.) et al., Ce11, 7. 279 (1976)), this 3' hairpin structure can serve as a primer for complementary DNA strand synthesis. The synthesis of this complementary strand is carried out by DNA polymerase II in a reaction mixture containing deoxynucleoside triphosphates.

H (Kleno Town I1.) et al., European Journal of Biochemistry (Eur, j, Bi
ochem, ), η, 37H1971)). The duplicated cDNA recovered by this technique has a 3' loop, resulting in the 3' hairpin structure of the single-stranded cDNA copy. This 3' loop is basically the method described by McCandliss et al., Methods in Enzymology.
It is cleaved by digestion with the enzyme S1 nuclease using the method of Yuzuru, 601 (1981).

The Sl nuclease degradation products are extracted with phenol-chloroform and the resulting cDNA is precipitated with alcohol from the aqueous phase.

A complete (i in ) corresponding to the human serum albumin gene
tact) double-stranded DNA (approximately 2000 base pairs), for example,
51) by sucrose density gradient centrifugation. To measure the size of DNA in sucrose density gradients, aliquots of density gradient fractions are electrophoresed in polyacrylamide gels with molecular weight markers. The resulting gel is first stained with ethidium bromide to visualize markers, followed by autoradiography to detect radioactive cDNA. Both portions of the density gradient containing DNA molecules larger than 1000 base pairs are pooled and the DNA is precipitated with ethanol.

Double-stranded cDNA prepared as above for propagation and selection
The A gene is inserted into a suitable loning vector used to transform a host cell suitable for boat fishing.

Suitable cloning vectors include a variety of plasmids and phage, although plasmids are preferred in this case.

WJJ for selecting a cloning vector includes its size, ability to replicate in host cells, presence of a selectable gene, and presence of a gene insertion site.

In terms of size, vectors are advantageously relatively small to allow insertion of large genes and to avoid directing large amounts of cellular nutrients and energy to the production of unwanted macromolecules. The vector also contains a complete replicon that maintains function after gene insertion. This replicon preferably controls the desired aspects of plasmid replication (eg, multiple copies of the cell or a single copy, or a controllable number of copies of the cell or the like). Genes specifying one or more phenotypic characteristics (preferably antibiotic resistance) facilitate selection of transformants. The insertion site is advantageously a unique restriction site for a restriction endonuclease. A cloning vector that meets all of these criteria is plasmid pBR322.

cDNA is conveniently inserted into this plasmid by the homopolymeric tailing method. The homopolymer tail is added to the 3'-water Bg of the human serum albumin double-stranded cDNA gene by reaction with the appropriate deoxynucleoside triphosphate in the presence of terminal deoxynucleotidyl transferase. The plasmid is cleaved by digestion with the appropriate endonuclease, and a complementary homopolymer tail is added to the 3'-hydroxyl group of the cleaved plasmid using the homopolymer tailing method. The optimal reaction condition is to add a dC residue to double-stranded cDNA (McCandli
ss, R, et al., p. 601, supra: Roychoudhury, R, et al., Nucleic A.
cids Research) 3,10] (197
6) and addition of dG residues to Pst I-treated pBR322 (Maeda, Nis (Maeda + S,
), Methods in Enzymology
in Enzymology)79°607 (19
81)). However, the preferred embodiment is 3'
Excess molar ratio of dXTP to terminal is 3000 to 50
The range is 00. The progress of the reaction is monitored until the chain length is approximately 15. Tailed cDNA and plasmids are recovered, for example, by phenol extraction followed by alcohol precipitation.

The homopolymeric ends of the two DNAs are complementary,
Anneal under appropriate conditions, H5A
Generating a recombinant plasmid containing the gene (Maeda, S.), Methods in Enzymology
79°611 (1981)).

Preferred strains of E. coli (E. coli) are basically Lederberg
) Law, Journal of Bacteriology (J, Ba
ctert-o1ogyH19, 1072 (1974)
transformed with this recombinant plasmid using
maintained indefinitely.

Typically, hundreds or thousands of clones are generated by these techniques and screened for the presence of the H3AI gene using, for example, rat serum albumin cDNA. Human-derived cD
Nick translated with 85% homology to NA (Maniats, T.
T, et al., Proceedings of the National Academy of Sciences
c, Nat'l Acad, Sci [ISA 72
．． 3961 (1975)) The rat-derived cDNA was transferred to a plasmid FCDNA attached to a nitrocellulose filter.
Grunstein, M. et al., Proc.
Acad, Sci USA) 72.3961
(1975); Southern.

Southern, ε0M, Journal of Molecular Biology (J, Mo1)
．． Biol, ), 98, 503 (1975)). In this technique, DNA from each colony (or group of colonies) is immobilized and denatured in discrete zones of a nitrocellulose filter. Instead, D.N.
A can also be electrophoresed in a gel before immobilizing it on the filter. A solution of radioactively labeled rat-derived cDNA is processed under hybrid conditions.

Unhybridized rat-derived cDNA is washed from the filter, and colonies containing DNA hybridized with rat-derived cDNA are identified by autoradiography. One positive clone was identified, but DN
A sequencing revealed that it was incomplete H5AC DNA. This H5Ac cDNA portion was nicked to rescreen the complete bank of clones.
nick translated
It was done. Thus 90 positive hybridizations
signal (hybridization signal)
)was gotten.

Positive clones were cultured in appropriate growth medium and plasmid D
Obtain large quantities of cells for NA extraction.

Plasmid DNA is extracted by conventional techniques (e.g., cell disruption, followed by phenol extraction and alcohol precipitation. Plasmid DNA and chromosomal DNA are extracted by, for example,
It is separated by electrophoresis or cesium chloride equilibrium centrifugation. Plasmid DNA containing inserts of approximately 1500-2000 base pairs is selected for further characterization.

After the cloned genes are excised from plasmid DNA, they are characterized by sequence analysis (Sanger F.
Sanger, F.) et al.
Op National Academy of Sciences Ninnisny (Proc, Nat'l Acad, 5c
iUSA 74.5463 (1977); Maxam, Nee.

A.) et al., Proc.
Nat'l Acad, Sci USA) 74,56
0 (1977)).

Prepro-H3A clones have been isolated by these techniques. E. coli (E. coli) HBIO transformed with a plasmid containing this prepro-H3Aift gene
I cultures were grown at the U.S. Department of Agriculture Northern Research Laboratory, Peoria, Illinois.

Department of Agriculture
Northern RegionalResearch
h Laborator NRRLNO, B-157
It has been deposited as No. 84. Partial restriction maps of this characteristic HSA gene insertion are shown in Figure 1 of the drawings and Figure 2.
The figure shows the amino acid sequence specified by the gene, along with the 5' to 3' linkage of the non-coding and coding regions.

The isolated gene consists of 2050 base pairs lacking the "tail". The gene is at the 5' end (base pairs 1-31)
and a non-coding region at the 3' end (base pairs 1858-2015). 5' end of the coding region (32-1
03 base pairs) is the 24 amino acid leader (leader
) (i.e., an 18 amino acid “ble” sequence followed by a 6 amino acid “pro” sequence) and a complete (m
a Lure) Human serum albumin protein is base pair 104
It is specified by the area from th to 1858th.

As used in FIG. 2 and elsewhere in the text, abbreviations have the following standard meanings.

A: Deoxyadenyl T: Deoxythymidyl G: Deoxyguanyl C: Deoxycytosyl Gly Nigericin Ala: Alanine Val: Valine Leu: Leucine 11e: Isoleucine Set: Serine Thr: Threonine Phe: Phenylalanine Tyr: Tyrosine Trp:) Liptophan Cys Nigistein Met: Methionine Asp: Aspartate Glu: Glutamate Lys: Lysine Arg: Arginine His: Histidine Pro Nibroline Gln: Glutamine Asn: Asparagine It is clear that due to the degeneracy of the genetic code, the nucleic acid sequence of the gene is subject to substantial variation. Will. For example, part or all of the gene can be chemically synthesized to provide a different nucleic acid sequence than that shown in FIG. However, if specific codon-amino acid designations are observed, the amino acid sequence will be preserved. Although the nucleic acid sequence of the human serum albumin gene and the amino acid sequence of the protein have been established, the gene of the present invention is not limited to a particular nucleic acid sequence, but includes all changes recognized by the genetic code.

The present invention was described using Escherichia coli (E, colt) as a host bacterium for recombinant DNA containing the H5A gene.

However, those skilled in the art of molecular biology will recognize that other Durham-negative bacteria such as Pseudomonas, Durham-positive bacteria such as Bacillus, higher unicellular organisms such as yeasts and molds, and It will be appreciated that mammalian cells can also be used for cloning and/or expression of the H3A gene.

The present invention will be further illustrated by the following examples, but the present invention is not limited thereto.

Example 1, Messenger RNA (mRNA) of H3A mRNA from ゛ was isolated from large liver tissue taken from a 10 year old accidental victim. Extra care was taken during the processing steps to prevent contamination of the mRNA samples with bonuclease. These procedures involve the use of novel sterile laboratory glassware, treatment of the solution with diethylpyrocarbonate if possible, followed by autoclaving, and preferably cryogenic treatment of the sample. Gloves were used to avoid contact between the sample and the skin.

Frozen human liver Fi1 tissue (10.5 grams) was
Virtis) homogenizer to add 210 ml of the solution (4M guanidine thiocyanate 10.1
M) Homogenized with Lis-H (1, pH 7, 510°IM 2-mercaptoethanol). Cell debris was pelleted by centrifugation in a Sorvall GSA rotor for 10 minutes at 4°C, 8750 rpm. The supernatant was transferred to a new centrifuge tube. supernatant, 0.04
volume of 1M acetic acid and 0. Five volumes of 95% ethanol were added. After 2 hours at −20°C, the mixture was heated to 750 rpm.
Centrifuged for 10 minutes at 4°C. The obtained pellets were 5
0ml cleaning solution (6M guanidine hydrochloride/1
0mM Naz EDTA, pH 7, 0/10m
Re-suspended in M dithiothreitol). 5500r
The fine particle debris pelleted by centrifugation at pm for 10 minutes and the supernatant were transferred to a new centrifuge tube. Add Ol to the supernatant.

4 volumes of 1M acetic acid and 0. Five volumes of 95% ethanol were added. After 2 hours at −20° C., the mixture was heated at 7200 r.
Centrifuged at pm for 20 minutes. The pellet was resuspended in 20 ml wash solution and 0.04 volume of IM# acid and 0.5 volume of 95% ethanol were added.

The mixture was kept at -20°C for 12 hours and then heated in a Sorval 1 SS 340-tar for 40 hours.
Centrifugation was performed at 8000 rpm for 10 minutes at °C. The pellet was resuspended in 15 ml sterile distilled water (dH20) and extracted with an equal volume of chloroform:butanol (4:1).

The aqueous phase was transferred to a new tube and 0.1 volume of 2.4M sodium acetate and 2.5 volumes of 95% ethanol were added.

After 2.5 hours at -20°C, RNA was pelleted by centrifugation. The pellet is 2mI! ! Resuspend in sterile distilled water. A total amount of RNA of 19°2 was recovered.

Next, the mRNA was converted into oligo(dT)・cellulose・
Affinity chromatography method (Av
iv) et al.: mentioned above and Pinus cantris (McCandl
iss et al.: previously described).

5 grams of oligo(dT) - Wash the cellulose column with 1 column volume of O, 1M sodium hydroxide,
All glue bonucleases present were denatured. It was then equilibrated with high temperature buffer (10mM Tris-HCl, p
H7,410, 5M NaC110, 5% sodium dodecyl sulfate (SDS)).

The total RNA sample dissolved in 1 ml distilled water was heated at 70° C. for 1 minute. Then cool to room temperature on ice, then cool to 0.
．． 1 volume of 5M NaC 1,0,0.04ml 0.5
M Tris-HCl 1 pH 7,5 and 0° 1 ml
0% sodium dodecyl sulfate (SDS) was added to the RNA. Then 8 ml of hot buffer was added to the RNA. The solution was then packed into the column at a flow rate of approximately 10 drops/min. After passing this solution through the column, unbound R
NA was washed and eluted from the column with hot buffer. Both fractions (1/2 ml each) were collected and 260 n of each fraction
The absorbance (A zho) of m was measured using a spectrometer. Column is A2. Washing was performed until the value of 0.05 or less.

The undesired RNA was further removed with a buffer solution at a low concentration (10
mM Tris-HCl, pl+7.410.2M NaCJ
The column was washed with 10.1% 5DS). Both parts are A
Samples were taken as described above until z6° dropped to 0.05.

Next, the mRNA was dissolved in elution buffer (10mM Tris-HC).
l, pH 7, 4/1mM EDTAlo.

The column was eluted with 1% 5DS). 1rr1 fraction,
Samples were collected until Az6° became lower than 0.05. The first 15 fractions (with the highest ODzbo values) are pooled and m, RNA is 2.4M with a volume of 0.1
It was precipitated by adding sodium acetate and 2.5 volumes of 95% ethanol and kept at -20°C for 12 hours. Next? The extracted mRNA was pelleted by centrifugation and
Resuspend in 0 μβ elution buffer. The resuspended pellets were heated to 70°C for 90 seconds, then cooled on ice and incubated at 0.5°C.
1 volume of 5M NaCl and 0.05 volume of 10% SD
S was added.

The eluted mRNA prepared above was then further purified by passing it through a second oligo(dT) cellulose column. A column packed with 0.1 gram oligo(dT) cellulose was washed with sodium hydroxide and then with the high salt buffer described above. The RNA was subjected to column chromatography and both fractions were collected as on the first column, with hot buffer, cold buffer, and elution buffer. The peak fractions obtained in the elution buffer step were collected, and the twice purified mRNA was precipitated and pelleted as before.

The mRNA was then subjected to particle size fractionation on a 12mβ sucrose gradient (McCandliss
) et al., Method in Enzymology (Metho
d in Enzymology)+79+pp,
56-58). A 5-20% sucrose gradient was created using gradient buffer (0,02M sodium acetate, pH 5,6).
and cooled at 4° C. for 3 hours. 100 of mRNA
Resuspend the µg in 100 µl of gradient buffer for 2 min.
Heated at 0°C. It was quenched in a water bath and then loaded at the top of the gradient. second 5-20% gradient, E. coli 16S and 23S rRNA as molecular weight markers (total amount 1010
Loaded with 0cr. The two gradients are Beckman SW40
38°000rpm with rotor 12. 5 hours 4℃
Centrifugation was carried out under the following conditions. Then, approximately 0.5 ml of both portions were collected and A44. (Fraction #1, for which we want to measure #1, is taken from the bottom of the gradient tube.) The A26O peak was divided into six groups A to F as shown in FIG. Pool the fractions of each group,
0.1 volume of 2.4M sodium acetate and 2.5 volume of 9
mRNA was precipitated with 5% ethanol.

mR encoding a protein of estimated size in ISA
Both groups containing NA contained rabbit reticulocyte lysate (Iysate) supplemented with 35s-methionine.
Identified by in vitro translation with a kit (provided by Bethesda Research Laboratories and used according to the manufacturer's instructions). The reaction mixture of each subgroup was 12
．． Contains the necessary compositions for translation of mRNA into radioactively labeled fluorescent matter detected by electrophoresis on a 5% polyacrylamide/SDS gel and subjected to fluorography.

The fluorogram showed a prominent protein band in the translation products of both subgroups B and C, estimated to be ISA (68,000 Daltons) in size. Group B contains a very low proportion of protein products in the unfavorable low molecular weight range, so the mRNAs in group B contain cD
was selected for use as a template for the synthesis of NA.

Example 2 H3A cDNAO In general, cDNA synthesis is carried out using pine cantris (McCa
ndl 1ss) et al. [Methods in Enzymolog
y), 79. PP, 601-607 (1981)
) was used. The use of radioactively labeled deoxynucleotides allows monitoring of the synthesis and calculation of products at each step.

The first strand of cDNA was synthesized using oligo-dT as a primer and mRNA as a template as shown in the following composition.

〉0.5 M pre-mixed and kept in water
l-Lis-H (1, pH 8, 320 μ! 1.4 M
MCI 10μlO, 25
M M g C! l z 8μ
lO,05M dATP, pH7,o
2μβ0.05M TTP, pH7,o
2μβ0.05M dCTP, pH7,
02μ10.05M dGTP, poy, o
2μ10.01M dithiothreitol
4μ! Sterile distilled water 4
5 μl water-soluble label, α”P-dCTP (10 μCi/
μβ〕f 100μl and the following composition was added〉 Oligo(dT) +z-+* (250# g/m
20 μm actinomycin D (500 μg
/ m'! + water soluble) 16μ! 10 μl mRNA, “B” fraction 201
! Sterile distilled water 37μl*A
MV reverse transcriptase (16U/μl) 7 p 1 Satoshi amount =
200 μl *Avian miniloblastosis virus (8vian myelobl)
ostosis virus/AMV) reverse transcriptase is
Since it is kept at -80°C, it will be dissolved for a while and then added as the final addition.

The reaction mixture was kept in water for 5 minutes and 2 μl was transferred and counted in ASC scintillation fluid to determine the specific activity of dCTP. The reaction mixture was then mixed with 46
Incubate for 10 minutes at °C. 0.2 of 20μl
M EDTA pus.

The reaction was stopped by adding O, and the mixture was then extracted with an equal volume of phenol:chloroform (1:1).

0.14 volumes of 80% glycerol were added and the sample was chromatographed on a 0.7×17 cm Sephadex G-100 column.

- After applying the sample to the column, add c100 buffer (1
0mM) Lis-HCC pH 8, O/1m, ME
DTA/100mM NaCl) was developed on the column and 5 drops (approximately 275 μl) of fractions were collected. The radioactive fraction was determined by Cerenkov counting (“Cerenkov
The cDNA fractions with the highest counts per minute were pooled.
/c DNA hybrid was prepared by combining O0 volume of 2.4 M sodium acetate and 2.5 volume of 95% ethanol.
- and placed in a dry ice/ethanol bath for 30 minutes, then at 4°C for 20 minutes.

A pellet was obtained by centrifugation at 10,000 rpm to obtain a precipitate. Pellets were collected in 300 μl of 0. 1M
The RNA was hydrolyzed by resuspending in sodium hydroxide solution and heating at 70°C for 20 minutes. -The terminal strand cDNA was left intact. The solution was made neutral by adding 30 μβ of LM HCl1. The DNA was prepared by adding 5MgtRNA, 1/10 volume of 2.4M sodium acetate and 2.5 volumes of 95% ethanol, placed in a dry ice-ethanol bath for 10 minutes, and placed in a microfuge for 10 minutes at 4°C.
refuge).

The pellet was resuspended in the following mixture.

40μit O, 5M KzSO4pl+7.
48μZ 0.25M MgC1゜2μ10.
1M dithiothreitol 1 μl O, 05M
dATP, pH 7, 01 μm 0.
05M dCTP, pH7, 01μm
0.05M dGTP, pH7, 01μf
O,05M TTP, pH7,01
24 μN Sterile distilled water 178 μl Next, 22 μl of DNA polymerase
ennow) fragment (5μ/μl, Boehringer
(Courtesy of Mannheim) was added.

The reaction mixture was then incubated in a 15°C water bath for 12 hours. 0.2μl of 20μβ DT A
pl+8,0 was added to stop the reaction.

The mixture was extracted with an equal volume of phenol:chloroform (1:1). 0.14 volumes of glycerol were added to the aqueous phase.

sample, which now contains two strands of McDNA.
was applied to a Sephadex G100 column, and the peak cDN
The A fractions were pooled and precipitated as before. double strand D
NA has a 3′ hairpin loop as mentioned above,
It was removed with 81 nuclease as follows. The pellet was resuspended in 72 μl of sterile distilled water. Then, 18ttl of 5-fold diluted S1 buffer (IM Na
(110,25M sodium acetate pH 4,515mMZ
n5o4/2.5% glycerol) was added.

The enzyme mixture contained S1 nuclease (20 μg/μm)2.
5 μl (50 units) was added to 47.5 μl Sl buffer. Then add 10 μl of the enzyme mixture to 90 μl
DNA? Yong? It was cooked in the evening and incubated at 37°C for 20 minutes. The reaction was stopped by the addition of 20 μm 0.2 μl DTA Na, and the reaction mixture was extracted with an equal volume of phenol:chloroform (1:1). The aqueous phase is
5-25% sucrose gradient, 38.00 Or
pm 17. Ultracentrifugation was performed at 5° C. for 5 hours.

Collect 1 ml of both aliquots and
kov) counting. Three pools were collected: fractions 1-6, 7-9° and 10-12. Fraction #1 was
Both portions were taken from the lowest end of the gradient. DNA was precipitated by adding 0.1 volume of 2.4 M sodium acetate, 1-2 μg of tRNA, and 2.5 volumes of 95% ethanol to each pool and incubating at -20°C. The DNA was centrifuged at 25 K for 30 minutes at 4°C.
Pelleted. After slightly dehydrating the pellet, the DNA from each pool was resuspended in 200 μβ of distilled water and again precipitated with ethanol and sodium acetate. The pellet was resuspended in 22 μl distilled water and centrifuged in a microfuge for 5 minutes to pellet insoluble material. 2 μl of each cDNA-containing supernatant was electrophoresed on a 6% polyacrylamide gel and analyzed. Gel autoradiography showed that the DNA in the pool of fractions 1-6 had an average size of 1100 bp (base pairs) and contained a 2oobp range of DNA. And this pool is filled with mancantoris (McCan).
dl 1ss) et al., supra 601 and the following pages).
cDNA by olimeric tailing method
It was used to add polyC tails to the 3' end of the protein. For this purpose, we have found that using 5000 molar excess of dCTP yields good results.

The reaction mixture is as follows.

20 μl cDNA (about 43 ng)-3HdCTP
(645μmo+, freeze-dried) 2.4μN 10-fold diluted TdT buffer *1.6μl Distilled water 24.0μl *10XTdT buffer = 1.4M cacodylic potassium 10
．． 3M) Lis-HC1, pH 7, 0/10mMCoC
Ilz/1 mM DTT reaction mixture at 37 °C
The cells were incubated for 2 minutes and 2 μl was taken for use in counting. Then 2 μl (6,66 units) of P-
Terminal deoxynucleotidyl transferase from L Bio Chemicals was added and incubation at 37°C continued for 5 minutes. Counting by 3HdCTP incorporation indicates that the 3' end of the cDNA has an average length of 14
It was shown to carry a "polyC tail" of nucleotides. 80μiT, E, +71 buffer (10mM) Lis-H
C1, pH 7, 6/1mM EDTA) was added to the DNA and the solution was extracted with an equal volume of phenol:chloroform (1:1).

Furthermore, the organic phase is 100μ! Reprocessed with distilled water and mixed the two aqueous phases.

The C-tailed double-stranded cDNA is then linearized with the restriction endonuclease Dan↓tI and then annealed to the "G-tailed" plasmid pBR332 DNA by the homobolimelink tailing method. The complementary single tAC and G “tails” are annealed,
A recombinant plasmid with cDNA inserted into the Ps±1 site was obtained.

200μ7! cDNA, C-tailed (39.2 ng
)10.5μI! pBR322-PstI,
G-tailing (302ng) 93μm Annealing buffer diluted 10 times* 626.5μ! Distilled water 930μ! The reaction mixture was placed in a separate water bath at 70°C, then the water bath was transferred to a 37°C room and gradually cooled to 37°C over the night. It was then transferred to room temperature, whereupon the water bath was cooled to 30° C. over several hours.

Additionally, the reaction mixture was stored at 4°C.

*(10x annealing buffer = 1.5MNa (1!
/100mM Tris-HCl.

pH 7, 5/10mM EDTA) E, Col HBIOI cells were adapted for transformation by known calcium chloride treatment methods. 200 μl aliquots of adapted HBIOI cells (aliq
uot) were each mixed with 40 μl of annealing reaction mixture and placed in water for 20 minutes.

Next, a heating treatment was performed at 42° C. for 2 minutes.

2.8ml Luria broth
was added to each tube and incubated at 37°C for 1 hour.

The contents in these tubes were recoated (1/2 ml aliquots) into tubes containing Soria broth supplemented with 0.7% agar and then poured onto Luria broth agar plates containing 25 μg/ml tetracycline. It was then incubated at 37°C until colonies appeared. (cD
Only cells transformed by pBR322 (with or without an NA insertion) can grow on tetracyclytic plates. Approximately 2500 transformed colonies grew on the plates.

Example 3 Transformants of total H3A cDNA were initially transfected with rat serum albumin (R3A
) cDNA fragments were screened.

The R3Ac cDNA fragment was obtained from the pBR322 plasmid containing 2000 pb of R3Ac DNA insert. This recombinant plasmid was obtained from the Department of Proceedings of the National Academy of Sciences Nynisny (
Proc, Nat'l Acad, Sci.

), similar to the plasmid p rAf b I described by Yukai 4370 (1979), but with a longer c
Carrying a DNA insert.

The 1480bp rat serum albumin (R3A) fragment is
Restriction endonuclease DanSat ± EI [R3Ac cDNA
(all uniform endonucleases used in this example were used by the method described in the manufacturer's instructions)
.

The fragments were then subjected to the “nick translation” method (Maniatis et al., PNAS USA;
72:3961 (1975) with α1Zp.

10 mj of approximately 80 individual transformants! ! culture
(culture) and plasmid DNA
A was isolated by the known plasmid "mini-pretub method". Partially purified plasmid DNA was electrophoresed on a 0.8% agarose gel. The DNA was analyzed using the “Sachin blotting method” (Southern, EM).
ern, E. M.) Journal of Molecule
Biology (J, Molec, Bio-1ogy)
98.503 (1975) from the gel to a nitrocellulose filter.

The nitrocellulose filter was filled with prehybridization solution (50% formamide 15-fold diluted SS).
C*/ 0, 05 M Na 3P Os.

pH 6, 515 times diluted Denhardt's 5t
ock*/100, czg/m1 salmon sperm DNA) for 2 hours at 42°C. The filter was mixed with a hybridization solution (50% formamide/10% dextran sulfate/5-fold diluted solution of SSC/20mM Na
, P O, , pH 6, 5/I XDenhardt's
5tock (150 μl/ml salmon sperm DNA).

The 1480 bp R3A fragment prepared by nick translation as described above was heated at 100°C for 5 minutes, then rapidly cooled in a water bath, and this probe was 2XlO' cpm of probe per ml of combined solution was added to the hybridization solution.

Filters were then incubated in hybridization solution for 18 hours at 42°C and then washed twice with 2:2 diluted SSC and once with O,1X5SC at room temperature.

Autoradiography of the filters revealed specific hybridization of the probes to all plasmid DNA.

*50 XDenhardt's 5tock= 1%
Polyvinylpyrrolidone/1% Ficoll/1% Bovine Serum Albumin (BSA) *SSC=150mM NaC4/15mM Sodium Citrate, Type 8 to pH 6,13 with citric acid Therefore, some Southern Plot filters
SS diluted 2 times at various temperatures from 65°C to 80°C
Washed with C. DNA from one plasmid on the filter washed at 65°C hybridized strongly with the probe.

DNA sequencing results in a “positive” test called 6C3
The clone was shown to be a partial length human serum albumin clone. Plasmid DNA was extracted from 6C3 culture+
(Culture) and digested with restriction endonuclease.

One of the resulting H3Ac cDNA fragments, approximately 475 bp in length, was isolated and "nick translated" for use as a probe.

For the entire bank of approximately 2500 clones,
This probe was screened using a modification of the hybridization method described by Grunstein et al., supra.

Transformed colonies were treated with 0.2% glucose and 25 μg
/ ml Tetracytalin plus Luquapros (Lu
ria broth) were separately removed from the plate into separate wells on a 96-well microtiter plate.

It was then incubated overnight at 37°C. Using a conversion device with 48 metal prongs, the samples in each medium were transferred to two Luri broths.
a broth/agar/tetracycline plates, one plate previously covered with a nitrocellulose filter. It was then incubated at 37°C for 2 days. The filters were then placed sequentially on Whatman filter paper soaked in one of the following solutions:

0.5M sodium hydroxide; 1M Tris, pH 7,
4; 1M Tris, pH 7,4; 2-fold diluted SSC;
90% ethanol, and 90% ethanol (in that order for 7 minutes per solution).The nitrocellulose filter was then heated in vacuo at 80° C. for 2 hours.

The prehybridization and hybridization methods were as described above except for washing three times at room temperature. Ninety positive hybridization signals were detected by autoradiography. “Positive clones” are further analyzed by restriction enzyme analysis (e.g. Pstl digestion).
and analyzed by the "Southern blot" hybridization method described above.

Clones with full-length H3Ac cDNA were identified and confirmed by DNA sequencing. This H3AcDN
The recombinant plasmid containing the A insert was named pGX401 and is shown in FIG. A partial restriction enzyme map of H3Ac DNA is shown in FIG. 1, and FIG. 2 shows the DNA sequence (5'-3' strand) of the cloned gene and the amino acid sequence that specifies it.

E, coltHBIOI transformed with pGX401
The sample was sent to the U.S. Department of Agriculture Northern Research Laboratory, Veolia, Illinois, as Reception NRRLNO, B-15784.
culture Northern Regional
Research Center).

[Brief explanation of the drawing]

Figure 1 shows full-length HS A c isolated by the method described in the text.
A partial restriction map of DNA clones is shown. Figures 2(a), 2(b), and 2(c) are full-length H3Ac in the order of (a), (b), and (c).
5' to 3' of the non-coding and coding regions of DHA
The amino acid sequence specified by the DNA sequence is shown along with the DNA sequence of the chain. FIG. 3 shows the A 26° profile of the sucrose density gradient fraction of mRNA. B group fraction is HS A
It was used as a template when synthesizing cDNA. Figure 4 shows pGX401. That is, full-length H3Ac DNA
The recombinant plasmid containing the insert is shown. However, the 5'Pst1 site was not regenerated. Figure 5(a) is a diagram showing the nucleotide sequence corresponding to the 95th to 102nd amino acids from the 5' end when the nucleotide sequence of the human serum albumin gene was determined directly from mRNA. human liver II
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Claims (1)

[Scope of Claims] 1. A human serum albumin gene comprising the following deoxyribonucleotide and amino acid sequences. [There is a gene sequence] Here, the 5' to 3' chain starts from the amino terminus, and each three-base chain indicates the amino acid it encodes. Additionally, the abbreviations have the following general meanings: A: Deoxyadenyl T: Deoxythymidyl G: Deoxyguanyl C: Deoxycytosyl X: A, T, C or G Y: T or C Z: When Y is C, A, T, C or G Y is T When is A or GH H: A, T, or C Q: T or A R: When Q is T, then C When Q is A, it is G S: When Q is T, it is A, T, C, or G Q When is A, T or CM M: A or G L: A or C N: When L is A, A or GL When L is C, it is A, T, C or G Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Cys: Cysteine Met: Methionine Asp: Aspartic acid Glu: Glutamic acid Lys: Lysine Arg: Arginine His: Histidine Pro: Proline Gln: Glutamine Asn: Asparagine 2, the human serum albumin gene according to claim 1, comprising the following deoxyribonucleotide sequence: [There is a gene sequence] Here, the 5' to 3' linkage indicates starting from the amino terminus, and the abbreviations are defined in claim 1. 3. A prepro-human serum albumin gene containing the following deoxyribonucleotide sequence. [There is a gene sequence] Here, the 5' to 3' chain indicates the amino acid encoded by each three-base chain starting from the amino terminus. Also, abbreviations are defined in claim 1. 4. The prepro-human serum albumin gene according to claim 3, comprising the following deoxyribonucleotide sequence: [There is a gene sequence] However, the 5' to 3' linkage indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. 5. The prepro-human serum albumin gene according to claim 3, comprising the following deoxyribonucleotide sequence: [There is a gene sequence. ] However, the 5' to 3' chain indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. 6. A plasmid having the ability to replicate in prokaryotes or karyophytes and carrying the following deoxyribonucleotide sequence encoding human serum albumin or prepro-human serum albumin. Deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] However, the 5' to 3' chain indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. Prepro-Deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] Here, the 5' to 3' chain indicates the amino acid encoded by each three-base chain starting from the amino terminal. Also, abbreviations are defined in claim 1. 7. A plasmid according to claim 6 carrying a human serum albumin gene or a prepro-human serum albumin gene comprising the following deoxyribonucleotide sequence: Deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] Here, the 5' to 3' chain indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. Prepro-Deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] Or, [There is a gene sequence. ] However, the 5' to 3' chain indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. 8. A microorganism transformed with a plasmid capable of replicating in prokaryotes or karyophytes and carrying a deoxyribonucleotide sequence encoding human serum albumin or prepro-human serum albumin. Deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] However, the 5' to 3' chain indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. Prepro-deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] Here, the 5' to 3' chain indicates the amino acid encoded by each three-base chain starting from the amino terminal. Also, abbreviations are defined in claim 1. 9. The microorganism according to claim 8, which is transformed with a plasmid carrying a human serum albumin gene or a prepro-human serum albumin gene containing the following deoxyribonucleotide sequence. Deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] Here, the 5' to 3' chain indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. Prepro-Deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] Or, [There is a gene sequence. ] However, the 5' to 3' chain indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. 10. Microorganisms are Escherichia (Escherichia)
The microorganism according to claim 8 or 9, which is a species of the genus Coli. 11. Microorganisms are NRRL No. B15784 (pGX4
The microorganism according to claim 10, which is substantially equivalent to 01). 12. In an aqueous nutrient medium containing absorbable carbon, nitrogen and essential mineral sources, growth factors, prepro-human serum albumin can be replicated in microorganisms under conditions that produce prepro-human serum albumin, and culturing a microorganism transformed with a plasmid having a deoxyribonucleotide sequence encoding albumin and recovering the prepro-human serum albumin so produced.
A method of producing human serum albumin. Prepro-Deoxyribonucleotide sequence encoding human serum albumin: [There is a gene sequence. ] Here, the 5' to 3' chain indicates the amino acid encoded by each three-base chain starting from the amino terminal. Also, abbreviations are defined in claim 1. 13. The method according to claim 12, wherein the deoxyribonucleotide sequence encoding prepro-human serum albumin is as follows. [There is a gene sequence. ] Or [There is a gene sequence. ] However, the 5' to 3' chain indicates that it starts from the amino terminus, and the abbreviations are defined in claim 1. 14. Microorganisms are Escherichia
14. The method according to claim 12 or 13, wherein the method is a genus Coli sp. 15. Microorganisms are NRRL No. B15784 (pGX4
15. The method of claim 14, which is substantially equivalent to 01).