JPS623796A - Production of interleukin 2 - Google Patents

Abstract

PURPOSE:To produce interleukin 2 out of a microbial cell, by introducing a plasmid having the DNA of signal peptide and the interleukin 2 gene linked to said DNA into a bacterial cell of Bacillus genus, and culturing the bacterial cell. CONSTITUTION:A plasmid containing the signal peptide DNA and the interleukin 2 gene linked to said DNA (e.g. pTY-amyIL2B42 obtained by integrating the interleukin 2 gene into plasmid pTUB285) is introduced into a cell of Bacillus genus bacterium, and the bacterial cell (e.g. Bacillus subtilis Marburg strain) is cultured. Interleukin 2 is produced and accumulated out of said bacterial cell.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a method for producing interleukin 2 (hereinafter referred to as "IL2J").IL2 has great potential to be used as a therapeutic agent for immunodeficiency, tumors, etc.

BACKGROUND ART As one method for producing IL2, there is a known method of culturing E. coli containing a plasmid into which a gene encoding IL2 has been inserted (European Patent Application Publication No. 00915
No. 39). However, in all microorganisms bred using conventional recombinant DNA methods, IL2 is produced and accumulated within the cells.
In order to recover IL2, complicated steps were required, including cell disruption and separation of IL2 from cytoplasmic protein.

Problems to be Solved by the Invention Therefore, it is an object of the present invention to
The purpose of this invention is to develop a method for producing IL-2 by producing L2.

Means to Solve the Problems In order to solve the above-mentioned problems, the present inventors cultured bacteria of the genus Bacillus into which a plasmid having the DNA of signal 41 and the IL2 gene connected to the same DNA was introduced. discovered a method for producing IL2 that does not involve producing and accumulating IL2 outside the cells of the same bacterium.

As a secretion vector DNA that functions in cells of the genus Bacillus, pTUB having a signal sequence of α-amylase is used.
226 (J.Biochem, 95, 8
7-93 (1984)), pTUB285 (G
ene, 34, 1-3 (1985)). The insertion sites of the IL2 gene into these vectors are described in the above-mentioned documents.

As IL2 gene, European Patent Application Publication No. 009153
All genes such as pIL2-50A described in No. 9 can be used.

A method for preparing recombinant DNA by connecting the above vector DNA and the IL2 gene can be, for example, the method of connecting using a synthetic NA linker as shown in FIG. 1, or other conventional methods.

The method of transforming Bacillus bacteria using the obtained recombinant DNA is also a conventional method. In other words, the Bacillus bacteria used as hosts include Bacillus subtilis, Bacillus subtilis,
Marperg strain, Batyas, Megatherium 1 Nochirus 0
Bacterial strains that can be used as hosts without modification or restriction systems are considered, such as Bacillus pumilis, Patylsus amyloliquefaciens, Bacillus rifeniformis, Bacillus plevis, and Bacillus stearothermophilum.

A method for transforming such a Bacillus bacterial host with the above recombinant DNA is the protoplast transformation method of Cohen et al. (Chang + S, + and Co
hen +S, N, , Mo1. Gen, Ge
netics, 168, 111 (1979)) and the competent cell method of Duncan et al.
an +C,H,* Wilaon lc,A, +-
and Young IF, E, eGene, 1
, 153 (1977)).

These methods are used to transform the IL2 expression plasmid, and the selectable marker trait on the vector DNA and the IL2 expression plasmid are transformed.
Transformants can be selected based on the presence or absence of production ability. Qualitative and quantitative analysis of IL2 can be carried out by the method described in GB 91539.

A method for producing IL2 using the obtained transformed strain, a Bacillus bacterium capable of producing IL2, involves using a microorganism capable of producing IL2 as a carbon source, a nitrogen source, and an inorganic ion.
Furthermore, if necessary, the culture may be carried out using an ordinary medium containing organic micronutrients such as vitamins and amino acids. The culturing method is usually carried out under aerobic conditions at a temperature in the range of 30°C to 40°C.

The method for collecting IL2yl cells from the obtained culture solution is to remove the bacterial cells by centrifugation, and then concentrate the culture supernatant by ammonium sulfate fractionation, etc., as described in British Patent Application Publication No. 91539. It can be done using the method described.

Example (1) Integration of IL2 gene into pTUB285 in the first step
This was done as shown in the figure. pIL2-5OA with restriction enzyme H
About 450 g produced by iAI and DraI cleavage
The bp fragments were separated and purified by 5% polyacrylamide gel electrophoresis. Next, this fragment was added to 33mM Tris-
acetate (P[(7,9), 66 mM potassium acetate, 10 mM h1lf2 magnesium, 0
．． 5mM dithiothreitol, 100μ dithiothreitol, 0.2mM dATP, dcTP, d
This was reacted with 4 units of 74 DNA polymerase (Zen Shuzo iR) at 37°C for 15 minutes in the presence of GTP and dTTP to form blunt ends. Separately, a Hlnd m linker called CCAAGCTTGG (manufactured by Zenshuzo Co., Ltd.) was added to 66 mMTr.
ls-HO2 (pH 7,6), 10 mM Mg (t2
．． 1mM ATP, 1mM spermidine hydrochloride, 15
T4 polynucleotide kinase (
It was phosphorylated by reacting with 3 units (manufactured by Zenshuzo Co., Ltd.) at 37°C for 1 hour. Subsequently, a phosphorylated linker was added to the blunt-ended fragment in the same reaction solution, and T4 DNA was added to the blunt-ended fragment.
This was carried out by reacting with 1.4 units of ligase (manufactured by Zenshuzo Co., Ltd.) at 4°C overnight. After the reaction was completed, the mixture was mixed with an equal amount of water-saturated phenol, and the aqueous layer was collected. The aqueous layer was extracted once with an equal volume of chloroform, and J DNA was recovered from the aqueous layer by ethanol precipitation.

DNA in 10 mM Tris-HCl (p) 17.5
) was dissolved in 7mM MgCl2.60mM NaC4, and 50 units of HIndI [[ was added thereto and reacted at 37°C for 4 hours to generate a cohesive end of HIndIII. After completion of reaction, phenol extraction, chloroform extraction,
After ethanol precipitation, v-linker removal was performed by 5% z +7 acrylamide gel electrophoresis.

Vector DNA was prepared as follows. pTUB28
The larger fragment generated after cutting 5 with HInd m (5,
3kbp) was separated and purified by 1.0% agarose gel electrophoresis.

This vector DNA and IL2 with the above linker added
The binding with the gene fragment is 20μ! 66mM Tris-
HCt (pH 7,6), 6 mM MgCl2.1
mM ATP.

This was carried out by adding 1.4 units of T 4 DNA ligase to a reaction solution of 10 mM dithiothreitol and reacting overnight at 4°C.

(2) Recombinant plasmid Bacillus sputum 207
-25 strain was introduced with S, Chang r S, N,
Cohen et al.'s protogellast transformation method (Mo1e,
Gen.

Genet4cm, 168, 111 (1979
)). Part f Rusu-x” Buchi, ps 207-25 stock 100rr
L/! After incubation for 2 hours at 37°C in pen assay medium containing 5JM and 10μ of lysozyme/0.5
M Shake Rose, 2.0rnM Maleic Acid, 20m
Suspended in MMgCt2 (hereinafter referred to as "S Yatsu"), 37
Cyprotoplasts were formed by incubating at ℃ for 1 hour.

Add 20 μl of the above ligated DNA-containing solution to 20 μl of the 2x 8MM solution and 0.5 μl of this protogellast solution.
Add d, and then add 40% PKG6000 1.5-, mix well, and leave at room temperature for 2 minutes.

5 m of SMM was added and protoplasts were collected by low speed centrifugation. The collected protoplasts were then suspended in SMM containing a standard concentration of in-assay medium.

By the way, 40% PEG 6000 solution is 40? PEG of
5WLLl for 6000! The concentration of SMM was adjusted to 100 with distilled water. After culturing protoplasts in the above medium at 37°C for 3 hours with shaking, 100μ
A plate of DI (3 medium) containing Kanamycin (0
, 8% agar, 0.5M Na succinate 2.0.5% Casamino acids, 0.5% yeast extract, 0.35%
Add 2HPO4,0,15%, PO4,0,5%
:ff-su, 0.02 MMgCt2.0.01%
The cells were spread on bovine serum albumin) and cultured at 37°C for 72 hours to regenerate. Since pTUB285 contains a gene that exhibits kanamycin resistance, only host bacteria that carry the plasmid will reproduce colonies. Regenerated colonies were randomly selected, the retained plasmids were isolated and purified, and a restriction enzyme cleavage test was performed to identify clones retaining the plasmid pYT-amy IL2B42 into which the IL2 gene had been inserted.

(3) Transformed strain harboring pYT-amy IL2B42 was treated with 0.5% yeast extract, 1.0%
The culture medium was shaken and cultured at 37°C in 100 mA' of poly4gtone, 0.54 NaCL, 0.2% glucose, sttt/ml kanamycin, pH 7.5, and the bacterial cells were removed by centrifugation. The supernatant was used as a sample for IL2 activity assay. Result: 10,000-15,000
Units/ml of IL2 activity was observed.

pTUB28s (see Figure 2) was constructed using the following procedure. pTUB 4 is B amHI of pUBllo
This is a plasmid in which a 2.3 kb DNA fragment containing the entire α-amylase gene is integrated. The entire base sequence of this integrated DNA fragment has already been determined (
Reference J, Bacteriol 156, 327 (
(1983)).

pUB110 is a plasmid that can be propagated within Bacillus bacteria and is widely used as a cloning vector.
- is being given. When pTUB4 is cut with EcoRI,
It is divided into 4.9 kb and 1.9 kb fragments. 4.9k
After making the fragment b blunt-ended with the Klenow 7 fragment of E. coli DNA J limerase, H1ndl
After adding a ll linker, the plasmid obtained by converting the T4 DNA' into ffl form with gauze is called pT.
It is UB201.

The next K 1.9 kb fragment was cut with AluI and α
- Isolate and purify a 424 bp fragment containing the amylase promoter and signal sequence, and add it to Hl n
H1 of the above pTUB201 by adding a d m linker
The plasmid obtained by integrating into the ndu site is pTU82.
It is 18. pTUB 218 was partially digested with HlndII, then blunt-ended with E. coli DNA polymerase I Klenow fragment, circularized with T4 DNA IJ gauze, further cut with Hlndm, and then cut with HlndIII from pKTH74. pTUB 285 is a plasmid that confers the ability to produce β-lactamase when the 1 to 4 kb fragment is introduced into a Bacillus subtilis marpark strain. pK
TH74 is the part of the β-lactamase gene encoded by pBR322 that lacks the signal sequence.
AAGCTGC Hlndl [via linker, p
It was incorporated into the Hlnd ll [portion of BR322 (Reference Proc, Natl.

Acad, Sci, 79, 5582 (198
2)).

As mentioned above, pTUB 285 is pUBllo's Bam
All base sequences have been determined at the HI site2.
This is a plasmid into which a 3 kb foreign DNA fragment has been inserted, and which can proliferate in bacteria belonging to the genus Gutilus.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the construction process of a plasmid having the interleukin 2 gene of the present invention. FIG. 2 is an explanatory diagram of the plasmid vector pTUB 285 used in the present invention. Patent application Ajinomoto Co., Ltd. Figure 1 IL2 ζON^ i H91AI/ Ora+

Claims (2)

[Claims] (1) Cultivating a Bacillus bacterium into which a plasmid containing the interleukin 2 gene connected to the signal peptide DNA is introduced, and producing and accumulating interleukin 2 outside the cells of the bacterium. Characteristic manufacturing method of interleukin 2. (2) The production method according to claim 1, wherein the signal peptide has the following amino acid sequence. [There is an amino acid sequence]