JPS60232091A - Complementary dna prepared from messenger rna - Google Patents

Abstract

PURPOSE:To obtain a complementary DNA capable of producing human B cell growth factor by a yeast or coliform bacillus, etc., by preparing the complementary DNA with a messenger RNA fraction obtained from a human T cell. CONSTITUTION:A complementary DNA (cDNA) obtained by separating a fraction containing a messenger RNA corresponding to human B cell growth factor polypeptide from a human T cell, and isolating the aimed cDNA corresponding to the human B cell growth factor messenger RNA (BCGFmRNA). The above- mentioned BCGFmRNA can be obtained by separating a fraction having 11S- 28S, preferably 15S-about 21S sedimentation constant range in sucrose density gradient centrifugation. The aimed cDNA is prepared by synthesizing single stranded cDNA complementary to the mRNA with a reverse transcriptase in the presence of dATP, dGTP, dCTP and dTTP using the BCGFmRNA as a template and an oligo dT as a primer.

Description

[Detailed Description of the Invention] This invention relates to metsenger RNA (hereinafter referred to as r mRNA).
It is written as J. ) well-prepared complementary DNA (r
It is written as cDNA J. ), please refer to Hirohi for further details) B
mR corresponding to a polypeptide with cell growth factor activity
Concerning eDNA prepared from NA.

h) B cell growth factor (hereinafter referred to as r BCGF J)
A polypeptide that is produced by T cells and promotes the proliferation of activated B cells sensitized by an antigen, and is necessary for antibody production by activated B cells. Therefore B
CGF may be used as a therapeutic agent for immunodeficiency diseases, tumors, etc. It is conventionally known that CGF is produced by human T cells.

In contrast, the present inventors found that mRNA corresponding to BCGF polyglutide (hereinafter r BCGFm
It is written as RNA J. ), and used this to successfully obtain a cDNA library corresponding to BCGF mRNA from the same fraction. This opened the door to producing fi BCGF using yeast, Escherichia coli, and the like.

The human T cells in the present invention are obtained from human peripheral blood, spleen, lymph nodes, tonsils, etc., and include both normal cells and malignant cells. For example, in the case of human peripheral blood T cells, human normal T cells can be obtained by subjecting human peripheral blood to density centrifugation using Ficoll-Paque to obtain phosphorocytes. Human T cells can be obtained by the E-rosette method using these lymphocytes and sheep red blood cells.

To obtain T cells from tonsils, loosen tonsil tissue with a bottle set in RPM11640 medium to obtain a cell suspension.
This is how to obtain a cylindrical sphere using the Schiffecol Pack's specific gravity centrifugation method. From this, human T cells can be obtained by the E-rosette method or the like.

Human malignant T cells include T cells isolated from T leukemia patients, T cells transformed by viruses such as ATLV and HTLV, normal human T cells, bone marrow lung cells, and malignant T cells. cell fusion strains, drugs.

This includes strains in which normal cells have mutated and become malignant due to radiation, etc.

A specific example of malignant T cells is CCRF-CEM.

(ATCC, CCL 119, Cancer 18
: 522-529. (1965)).

HPB-MLT (Int, J, Cancer, 2
1166 (1978)), HPB-ALL (Int.
J. Cancer, 21, 166 (1977) =
) , , TALL (Nature, 267, 8
43 (1977)), RPMI-8402 (J, Na
tl.

Cancer In5t, 55+11 (1975))
and so on.

In order to culture these human T cells to produce BCGF mRNA, human T cells are cultured in a conventional medium using a conventional method, and preferably the culture is continued until BCGF activity begins to be produced in the culture medium. Just keep going.

The medium used is Rosewell Park Memorial Institute 1640 medium (Rosew
ell Park Memorial In5tltu
tsl 640 + hereinafter abbreviated as RPMI 1640) is suitable, but Dulbecco's modified Eagle's medium (
Dulbecco's Modif led Eagle
e Medium) + Eagle basal medium (Eagl
e+sMinimum Es5nential Medi
Known cell growth media such as C1ick medium can be mentioned. These media include
It is used by adding 10% fetal bovine serum (hereinafter abbreviated as FBS), newborn bovine serum, horse serum, or human serum. If an appropriate growth-promoting substance is used, serum may not be necessary in some cases. For example, BCGF mRNA can be produced using RITC serum-free medium containing 0.51 BSA and bovine serum albumin, or RITC serum-free medium containing no BSA. 1 When using normal T cells, it is necessary to add a BCGF production attractant to the medium, but when using malignant T cells, BCGF mRNA is often produced even without adding the attractant. Sometimes, BCGF mRNA is not produced in a medium that does not contain a BCGF production inducer, but by adding a BCGF production inducer, BCG
Some strains even begin to produce FmRNA. Also, BCG
Even for strains that produce BCGF mRNA in a strong medium with the addition of an F production inducer, adding a BCGF production inducer to the medium usually does not reduce the BCGF mRNA production ability, and sometimes increases the BCGFmaNA production ability. Sometimes.

As a BCGF production inducer, ConA + PHA
and forsol esters such as PMA.

These are added to the medium alone or in combination.

The BCGF-producing caustic substance has a cell density of 1 to 5 x 10' cells/
It is suitable to add it to the cell fluid of d or more.

Cultures are usually 35 to 50 cells/- at a cell density of 1 to 5 x 10 cells/-.
It is carried out at 38° C. in a stream of 4 to 6 carbon dioxide gas. Culture is B
It is most desirable to continue until several hours before or after the time when CGF activity is found in the culture medium, but it may be continued until after that, as long as BCGF mRNA is found in the cells.

To separate the obtained T cell BCGF mRNA or an RNA fraction containing it, sucrose density gradient centrifugation, gel electrophoresis, adsorption column chromatography, etc. can be used.

BCGF mRNA has a sedimentation constant in the range of 118 to 288 in sucrose density gradient centrifugation, especially 13S to 26S1.
Furthermore, it is particularly present in mRNA fractions corresponding to molecular weights around 15S and around 218.

To test whether BCGF mRNA is contained in the separated mRNA fraction, the mRNA may be translated into protein and its physiological activity examined, or the protein may be identified using an antibody or the like. For example, the African claw, which is a system commonly used to translate mRNA into protein, is introduced into oocytes of Xenopus 1aevis.
Translation by injecting RNA or Reti-cu
locyte-lyzate + reticulocyte lysate,
Translation into protein is performed using a cell-free system such as Wheatgerm.

The produced protein is examined for BCGF activity using the method described later.

Using the obtained RNA fraction containing nine BCGF mRNA,
Prepare cDNA. That is, using mRNA as a template,
dATP, dGT as oligo dTfcf primers
P, dCTP.

Single-stranded cDNA complementary to l) mRNA is synthesized using reverse transcriptase in the presence of dTTP, and template mR is synthesized by alkaline treatment.
After degrading and removing the NA, double-stranded cDNA is then synthesized using reverse transcriptase or DNA polymerase using the single-stranded cDNA as a template.

By inserting the cDNA thus obtained into an appropriate vector DNA and inserting it into the host cell of the vector DNA used, B
Probes used to obtain DNA clones encoding the CGF ytP repeptide can be obtained, and furthermore, BCGF can be produced by transformed cells.

More specifically, for example, both ends of the obtained DNA are treated with exonuclease if necessary, appropriate NAs are connected to each end, or a plurality of bases in an annealing-enabled combination are polymerized.

Thereafter, this is incorporated into a microbial vector. The method of integration involves cleaving the vector with an appropriate restriction enzyme, and polymerizing a suitable linker or a plurality of annealing-capable combinations of bases, if necessary. Double-stranded DNA processed in this way
and vector DNA, and connect them using suga-ze.

The obtained recombinant DNA is introduced into a vector host microorganism. Suitable host microorganisms include microorganisms of the genus Escherichia such as Escherichia coli, microorganisms of the genus Bacillus such as Bacillus subtilis, and microorganisms of the genus Satucharomyces such as Satucharomyces cerevisiae. Vectors used for these microorganisms are illustrated below. (Refer to Protein Nucleic Acid Enzyme Vol. 26, No. 4 (1981)) p, sc'lo1, p of EK system Grasmid-covered (stringent type)
RK353. pRK646゜pRK248, pDF
41 etc., EK-based plasmid vector (relaxed type)
Ca1E1, p■51. pAc105°R8F21
24, I) CRI, pMB9, BR313
, pBR322, TIBR324, pBR325, pB
R327, pBR328, pKY2289.

pKY2700, p)G'J80, pKC7,
pKB 158, pMK2004.

pACYC1, pACYC184, λdu1, etc., λgt
λgt・λC, λgt・λB, λ of system phage vectors
■S・λC2λWES ・λB'λZJvir−λB/
, λALO−λB, λWEST Ts 622 , λDa
m, etc., Sharon 4A of Sharon Vector, Sharon 3A
.

Sharon 16A, Sharon 13A, Sharon 14A, Sharon 15A, Sharon 8. Sharon 10. Sharon 1
7. Sharon 20 etc., Tiollaia
) Glue-7' vector 4) L512, λZEQS'
, λZYV 5Φ, λZUVΦ2゜λZUVΦ3. λ
YEQSΦ1. λYEQSΦ, λYEQSΦ3. λBa
m + λSst, etc., Bacillus subtilis fluorasmid vector pT
A 1015.

pLs45, pTA1020, pLs2B
, pLs 13 , pTA1050.

pTA 1060, pTA 1030, pTA
1031, plasmid vectors derived from Staphylococcus p'r 127 , pc 194 .

pc221, pC223, pUB112, pU
B110, psAO501.

psA2100, pE194, pTP4,
pTP5, etc., enzyme vector) pJDB□19. YEp□
3, YRp 7, Y□p 1, pyc,
p'rc 2゜Microbial vector, e.g. pBR3
227Th which PstI or EcoRI 5ite
The BCGF can be integrated into a site according to the purpose and transformed into an appropriate host to express the BCGF in the host.

The BCGF activity assay method used here is as follows.

That is, 50 μl or 25 μl of the sample is added to the wells of a 96-well microtiter plate, and the total volume is made up to 100 μt with RPM11640 medium containing 10 g of tallow serum and 5×10 M of 2-mercagtoethanol. 2. B cells prepared from mouse spleen cells were added to this.
Add 100μ to each well for a cell density of 5×10′ cells/100μt. 37℃.

After static culture for 64 hours in a 5-inch carbon dioxide incubator,
After adding 1 μCi of tritiated thymidine to each cell and culturing for 8 hours, the cells are harvested according to methods well known in the art, and the amount of radiation taken up into the cells is measured. Since a culture supernatant with high BCGF activity has a large amount of tritiated midine taken up into the cells, the amount of BCGF contained in the sample can be easily determined.

Example (1) Human T-T hybrido-muff 7A cells (Journal of Experimental Medicine Vol. 157, 5
83 (1983)) were suspended at a cell density of 5 x 105 cells/ml in RPM11640 medium 1000-ml containing 10 g of tallow serum, placed in a Falcon rotary culture bottle, and incubated at 37°C for 4 days. The cells were cultured and collected by centrifugation. These cells were suspended in the above-mentioned medium at a cell density of 2 x 10' cells/-, and placed in a Falcon rotary culture bottle at 1 cell density.
oooy and cultured with rotation for 12 hours.

(2) 77A cells obtained in this way (12Kari010
Cells) were suspended in 2001 nl of PBS solution, washed twice by centrifugation, and then treated with Ribonucleosides-Vanadyl C, a nuclease inhibitor.
R8B solution (10mM) containing oplex (10mM)
mM Tris-HCt, pH 7,5°10 mM N
aCt, 1.5 mM MgC12) 200 ml
suspended in.

Next, 1NP-40 was added to a concentration of 0.05%, stirred gently, and centrifuged for 5 minutes at 3.00 Orpm to remove nucleic acids.
Immediately after adding DTA (5mM), an equal volume of phenol was added to extract cytoplasmic RNA. After repeating the phenol extraction three times in total, the RNA was precipitated with 2 volumes of ethanol, and the precipitate was collected by centrifugation and added to 10 rnM Tris-H.
C.L.

It was dissolved at pH 7.5. The amount of RNA thus obtained from 77A cells was 100 μ.

Next, mRNA from a part of RNA (351111iF) with
Oligo(dT)-cellulose (P,
L, Bio-chemicals + Type 7
) was used to perform column chromatography. Adsorption was performed using 20 mM Tris-HCt + pH 7,5, 0,5
RNA was dissolved in a solution of M NaCl and 1mM EDTA. After washing with the above buffer, elution was performed with water and 10mM EDTA.
This was performed by alternately eluting mRNA with Tris-HCL (pH 7,5). As a result, the eluted m
The amount of RNA was 600 μg.

Furthermore, this mRNA was subjected to sucrose density gradient centrifugation (50mMT
ris-HC4+pH7.5, 1mM EDTA,
5-25% sucrose density gradient with 0.2 M NaC4, 35,00 in H3tachi PR840 rotor
0 rprn, 12 hours, 4°C) and fractionated at 12
~288 mRNAs were obtained.

(3) The mRNA fraction obtained here was added to one African frog oocyte according to the assay method described above.
When the oocyte culture supernatant obtained by injecting μg of thymidine by microinjection was subjected to a BCGF activity assay, the amount of tritiated thymidine was detected as shown in FIG.

There were some stains, and the mRNA of these fractions was
It was proven that it contains BCGF mRNA.

(4) Next, 12 containing BCGF mRNA obtained here
~28S mRNA J, cDNA was synthesized in vitro.

That is, 50 mM Tris-HC4 buffer (pH
7,5).

30mM NaCt, 6mM MgCl2.5
mM dithiothreitol (DTT), 0.5 m
M each dATP, dGTP, dCTP.

dTTP (dCTP includes P-labeled)
.

0.75μ9 oligo (dT) 1g, 9.6μ9m
RNA and 15 units of AN4%' reverse transcriptase (J,
W, Beard) and kept at 41°C for 90 minutes.

As a result, about 1.9 μg of single-stranded cDNA was synthesized. To remove mRNA from the reaction solution, add NaOH to the reaction solution.
Add the solution to 0.33 N NaOH and dilute to 1 at room temperature.
After leaving for 5 hours, the solution was neutralized and
-50J column. This resulted in 1.2μg of c
DNA was collected.

50mM phosphate buffer (P"7.5), 10r
nWi NaCt2゜10mM DTT, 0.7
5- each of dATP, dGTP, dCTP.

dTTP (dCTP includes those labeled with 3H).

1.2 μg single-stranded cDNA, 8 unit Polymerase I (BRL, USA)
were mixed and the reaction was carried out at 15°C for 15 hours. This reaction yielded 1.3 μg of double-stranded cDNA.

Then 50mM sodium acetate (p'14.5).

0, 2M NaCl, 1mM ZnCl2.1.3
After mixing μ9 double-stranded cDNA and incubating for 20 minutes at 37°C, 0.25 units of nuclease St (
Sankyo Co., Ltd.) was added, and the mixture was further incubated for 15 minutes. Thereafter, 1.2 μg of double-stranded cDNA was collected.

[Brief explanation of the drawing]

FIG. 1 shows an example of fractionation of Metsenger RNA of the present invention. Patent applicant Ajinomoto Co., Inc.

Claims (1)

[Scope of Claims] 1) and) Complementary DNA containing methsenger RNA corresponding to a polypeptide having B cell growth factor activity and prepared using a methsenger RNA fraction obtained from human T cells 2) Complementary DNA according to claim 1, wherein the Metsenger RNA fraction has a sedimentation constant in a certain range of IIS to 288t in sucrose density gradient centrifugation.