CN1113954A

CN1113954A - Producing technology for interleukin-6

Info

Publication number: CN1113954A
Application number: CN 94106607
Authority: CN
Inventors: 奚绍祁; 孙玉昆
Original assignee: Harbin City Medicine Engineering Tech Development Center
Current assignee: Harbin City Medicine Engineering Tech Development Center
Priority date: 1994-06-24
Filing date: 1994-06-24
Publication date: 1995-12-27

Abstract

The present invention utilizes the PCR technology to obtain the gene fragments of inter-leukin-6 (IL-6 for short) and introduces the second SD sequence, thus the semi synthesized IL-6 functional gene is obtained to construct the efficiently expressed IL-6 engineering bacteria which are cultured in a nutrient medium, the expressed inclusion body is separated from the engineering bacteria, after extrative renaturation, the IL-6 active protein is obtained. Said method is used to make the bioregulator IL-6 having obvious anti-cancer activeness and capable of increasing blood platelet, that is the high pure IL-6 is produced by the industriallization of gene engineering culture fermentation technology.

Description

Producing technology for interleukin-6

Disclosed by the invention is a kind of obvious anti-cancer properties that has, and the production method of the biomass cells factor of platelet increasing particularly makes method and the relevant purification step thereof of this biological activity protein by the engineering strain fermented extracted.

Interleukin-6 (Interleukin-6) is a very broad profile of cytokine of a kind of biologic activity, and many cells can produce interleukin-6 (being IL-6), comprise T cell, B cell, monocyte, circular cone parent cell, endotheliocyte etc.Mitogen and antigen can stimulate T cell and B cell to produce IL-6.IL-6 has obvious antitumour activity, and the biological immune of platelet increasing is regulated, and can be used for the treatment of tumour and hematopoietic disorder.The common method output of utilizing cultivator somatocyte or cell strain to produce IL-6 is very low, can't satisfy clinical trial or as the needs of medicine, and the present invention invents along with engineered projection and development.Make biological activity protein make this bacterial classification have IL-6 insert structure gene by the fermented extracted of bacterial classification.

The purpose of this invention is to provide a kind of structure in early stage, cultivate from bacterial classification, to the production process of protein purification, promptly a kind of industrial requirement that meets, cost is low, and high yield is arranged, and can be used for the production technique of the IL-6 of human body.

Technological process of the present invention comprises:

A utilizes round pcr, makes up the engineering strain of the interleukin-6 that efficiently expresses;

B modifies the base in above-mentioned bacterial classification 5 ' terminal functional gene section, and inserts second SD sequence, promptly makes up its expression vector;

The c fermentation, this bacterial classification of escherichia coli high-level expression.

This technological process also comprises the purification process with molecular sieve gel filtration and high performance liquid chromatography.

Aforementioned technological process a can change into: (1) is from cultivating U ₉₃₇In the cell strain, extract MRNA, be transcribed into CDNA; (2) design, synthetic oligonucleotide primer thing comprise that Hind III and Ndel enzyme in the five terminal derivateds cut the enzyme of the Xbal of position and three terminal primers and cut the position; (3) with U ₉₃₇CDNA is a template, and pcr amplification obtains the interleukin-6 gene fragment; (4) with gel electrophoresis with this fragment Separation and Recovery; (5) with this fragment cloning to PCR ₂₀₀₀Change DH in the carrier over to ₂₅Be subjected to thalline; (6) screen the hickie bacterium colony, enzyme is cut.

The base in above-mentioned bacterial classification 5 ' terminal kinetic energy gene section among the aforementioned technological process b is modified and can be comprised: (1) will be by translating 6 dna fragmentations of theoretical synthetic through T ₄Starch phosphorylase, ligase enzyme, electrophoresis separate, and obtain the dna fragmentation of 143 base pairs; (2) from PCR interleukin-6 carrier, cut, separate through Taq I/Xba I enzyme, obtain the dna fragmentation of 454 base pairs; (3) 454 base pair fragments are connected with the dna fragmentation of above-mentioned 143 base pairs, obtain 597 base pair fragments.

In the aforementioned technological process, the expression body of the interleukin-6 of structure is: (1) carrier PEX-2 cuts, separates through the BsrBI/SspI enzyme, obtains the big fragment of DNA; (2) remove the Amp gene, be connected with the Kan gene fragment and obtain PKEX-2; (3) PKEX-2 cuts through Hind III and Xba I enzyme, and the dna fragmentation with 597 base pairs is connected again, is transformed into DH ₂₅Be subjected in the thalline, cut with dna sequence dna through enzyme and identify, obtain positive colony PKEXIL-6; (4) forward the PKEXIL-6 carrier DNA to N _4830-1In the intestinal bacteria, obtain the industrial strain of interleukin-6.

Fermentation described in the aforementioned technological process: can change into this bacterial classification of escherichia coli high-level expression: spend the night positive colony PKEXIL-6 (1) at the 100mlLB culture medium inoculated; (2) be diluted in the fermentation culture with 1: 50, spend the night, when temperature reaches 42 ℃, induced 3-6 hour, receive bacterium, use the SDS-PAGE electrophoresis, dyeing 25-35 ℃ of jolting; (3) with the bacterial classification smudge cells, centrifugal, separate, with the throw out washing, centrifugal, obtain inclusion body.

Purification process described in the aforementioned technological process can change into: (1) will stir 0.8-1.2 hour with the solubilization of inclusion bodies of the interleukin-6 of escherichia coli high-level expression in guanidine hydrochloride solution, filtered with molecular gel, the SDS protein electrophoresis, collected main peak; (2) in buffered soln, dilute, make renaturation; (3) after hyperconcentration, use gel filtration chromatography, collect main peak, the high performance liquid phase purifying.

The invention has the advantages that:

1, produce with this method, output obviously improves, and with in the interleukin-6 of escherichia coli high-level expression, IL-6 albumen accounts for bacterial protein and is not less than 20%.

2, the IL-6 purity height of producing with this method, purified IL-6 purity is not less than 97%, and directly clinical application is in human body.

3, construct the engineering strain that efficiently expresses IL-6, make the IL-6 can suitability for industrialized production, for road has been opened up in its further clinical application.

4, the IL-6 cost of producing with present method is far below original experimental technique.

5, active high, the specific activity of final pure product is not less than 1 * 10 ⁸Unit/ml.

Fig. 1 is the IL-6DNA sequence chart;

Fig. 2 expresses structural map for PKEX-2 IL-6.

Do further comprehensive explanation below in conjunction with accompanying drawing:

Fig. 1 IL-6DNA sequence by synthetic IL-6 five terminal gene fragments (1-101 base), is formed with natural IL-6 gene fragment (102-558 base).Gained IL-6 protein structure is identical with natural IL-6 protein structure.

One, the clone of IL-6 function area gene

From cultivating U ₉₃₇In the cell strain, extract mPNA, be transcribed into CDNA design synthetic oligonucleotide primer thing then

Five terminal primers

CCA?AGC?TTC?ATA?TGC?CAG?TAC?CCC?CAG?GA

Comprise that Hind III and Nde I enzyme cut the position

Three terminal primers

CTC?TAG?ACT?ACA?TTT?GCC?GAA?G

Comprise that Xba I enzyme cuts the position

With U ₉₃₇CDNA is a template, and pcr amplification obtains the IL-6 gene fragment, with above-mentioned pcr amplified dna fragment, after gel electrophoresis separates, reclaims, directly is cloned into PCR ₂₀₀₀In the carrier, change DH over to ₂₅Recipient bacterium, screening hickie bacterium colony, enzyme are cut and the dna sequence dna calibrating, obtain the PCRIL-6 of positive colony.

Two, semisynthetic IL-6 gene fragment

By Computer Analysis, and the utilization optimization is translated theory, six segment DNA fragments below the present invention designs, synthesized

（1）AGC?TTG?GGT?ATT?AAT?AAT?GTA?TCG?ATT?AAA?TAA?GGA?GGA?ATA?ACA

（2）TAT?GTT?ATT?CCT?CCT?TAT?TTA?ATC?GAT?ACA?TTA?TTA?ATA?CCC?A

（3）TAT?GCC?GGT?TCC?GCC?TGG?TGA?AGA?TTC?TAA?AGA?CGT?TGC?TGC?T

（4）GGT?GCG?GAG?CAG?CAA?CGT?CTT?TAG?AAT?CTT?CAC?CAG?GCG?GAA?CCG?GCA

（5）CCG?CAC?CGT?CAG?CCG?TTA?ACC?TCT?TCC?GAA?CGT?ATC?GAC?AAA?CAG?ATC?CGT?TAC?ATC?CT

（6）CGA?GGA?TGT?AAC?GGA?TCT?TTG?TCG?ATA?CGT?TCG?GAA?GAG?GTT?AAC?GGC?TGA?C

Above-mentioned dna fragmentation is through T ₄After phosphatization enzyme, ligase enzyme, the electrophoretic separation, obtain the dna fragmentation of 143 base pairs.From the PCRIL-6 carrier, cut through Taq I/Xba I enzyme, separate the dna fragmentation that obtains 454 base pairs.Then, be connected with the dna fragmentation of above-mentioned 143 base pairs, and then obtain the dna fragmentation of 597 base pairs.

Three, make up the expression vector of IL-6

Carrier PEX-2 cuts through BsrBI/Ssp I enzyme, separates to obtain the big fragment of DNA, removes the Amp gene, is connected with the Kan gene fragment then, obtains PKEX-2, and PKEX-2 cuts through Hind III and Xba I enzyme, is connected with the dna fragmentation of above-mentioned 597 base pairs, is transformed into DH ₂₅Be subjected in the thalline, cut with dna sequence analysis through enzyme and identify, obtain positive colony PKEXIL-6, last, make the PKEXIL-6 carrier DNA forward N to _4830-1In the intestinal bacteria, and then obtain the IL-6 engineering strain.

Four, escherichia coli high-level expression IL-6

With above-mentioned positive colony, at 100 milliliters of LB substratum (10g/L Peptone, 5g/L Yeast Extract 5g/L Nacl, 20mg/L kantlex) after inoculation is spent the night, to be diluted to (5g/L Yeast Extract 5g/L glucose 7g/L K in the fermentation culture at 1: 50 ₂HPO ₄, 8g/L KH ₂PO ₄, 5g/L(NH ₄) ₂SO ₄, 1g/L mgSO ₄7H ₂O, the little molten metal of 3ml/L, 3ml/L vitamin mixture, 20mg/L kantlex).Spend the night 30 ℃ of following joltings, work as OD ₆₀₀When reaching 10-50,, induced 3-6 hour increase in temperature to 42 ℃, conventional receive bacterium after, with SDS-PAGE electrophoresis, dyeing.The thin layer chromatography scanner analysis revealed.IL-6 albumen accounts for the body total protein and is not less than 20%.

The bacterial classification of receiving, through French Pressarer under 5MPa, smudge cells or with the Ultrasonic Cell Disruptor smudge cells, centrifugal, separate, its throw out is after the plain liquid washing of 2M urea, and is centrifugal, obtains inclusion body.

Five, the sex change of IL-6 and purifying

Above-mentioned inclusion body held separate in 6M guanidine hydrochloride solution (0.1M Tris-HCL5mMEDTA/1mm DTT PH8.0) stirring at room 1 hour, filter through molecular gel, after the SDS protein electrophoresis is identified, collect main peak, then, be diluted in (0.1MTris-HCL PH8.0) buffered soln renaturation at 1: 10.This diluent is behind ultrafiltration and concentration, and last sample chromatography in another gel column is collected main peak, and after the high performance liquid phase purifying obtains pure product, pure product are by SDS-PAGE electrophoresis and HPLC calibrating, and purity is not less than 97%.

Six, determination of activity

The determination of activity of all samples, be with the strain of 7TD1 hybrid cell measure (Van Snick et al 1986 Proc Natl Aead Sci U S A 83: 9678-9683) the CL activity of final pure product is not less than 1 * 10 ⁸Unit/milligram.

Claims

1, a kind of production technique of interleukin-6, its technological process comprises:

C fermentation: with this bacterial classification of escherichia coli high-level expression.

2, the production technique of interleukin-6 according to claim 1 also comprises the purification process with molecular sieve gel filtration and high performance liquid chromatography.

3, the production technique of interleukin-6 according to claim 1 and 2, described technological process a can change into: (1) is from cultivating U ₉₃₇In the cell strain, extract MRNA, be transcribed into CDNA; (2) design, synthetic oligonucleotide primer thing comprise that Hind III and Ndel enzyme in the five terminal derivateds cut the enzyme of the Xbal of position and three terminal primers and cut the position; (3) with U ₉₃₇CDNA is a template, and pcr amplification obtains interleukin-6, gene fragment; (4) with gel electrophoresis with this fragment Separation and Recovery; (5) with this fragment cloning to PCR ₂₀₀₀Change DH in the carrier over to ₂₅Be subjected to thalline; (6) screen the hickie bacterium colony, enzyme is cut.

4, the production technique of interleukin-6 according to claim 1 and 2, the base in the above-mentioned bacterial classification 5 terminal kinetic energy gene sections among the described technological process b is modified and comprised: (1) will be by translating 6 dna fragmentations of theoretical synthetic through T ₄Starch phosphorylase, ligase enzyme, electrophoresis separate, and obtain the dna fragmentation of 143 base pairs, and (2) are cut, separated through Taq I/Xba I enzyme from PCR interleukin-6 carrier, obtain the dna fragmentation of 454 base pairs; (3) 454 base pair fragments are connected with the dna fragmentation of above-mentioned 143 base pairs, obtain 597 base pair fragments.

5, the production technique of interleukin-6 according to claim 1 and 2, its expression vector of described structure is: (1) carrier PEX-2 cuts, separates through the BsrBI/SspI enzyme, obtains the big fragment of DNA; (2) remove the Amp gene, be connected with the Kan gene fragment and obtain PKEX-2, (3) PKEX-2 cuts through Hind III and Xba I enzyme, and the dna fragmentation with 597 base pairs is connected again, is transformed into DH ₂₅Be subjected in the thalline, cut with dna sequence dna through enzyme and identify, obtain positive colony PKEXIL-6; (4) forward the PKEXIL-6 carrier DNA to N _4830-1In the intestinal bacteria, obtain the industrial strain of interleukin-6.

6, the production technique of interleukin-6 according to claim 1 and 2, described fermentation: be with this bacterial classification of escherichia coli high-level expression: spend the night positive colony PKEXIL-6 (1) at 100ml LB culture medium inoculated; (2) be diluted in the fermentation culture with 1: 50, spend the night, when temperature reaches 42 ℃, induced 3-6 hour, receive bacterium, use the SDS-PAGE electrophoresis, dyeing 25-35 ℃ of jolting; (3) with the bacterial classification smudge cells, centrifugal, separate, with the throw out washing, centrifugal, obtain inclusion body.

7, the production technique of interleukin-6 according to claim 2, described purification process is: (1) will be with the solubilization of inclusion bodies of the interleukin-6 of escherichia coli high-level expression in guanidine hydrochloride solution, stirred 0.8-1.2 hour, filter SDS protein electrophoresis, collection main peak with molecular gel; (2) in buffered soln, dilute, make renaturation; (3) after hyperconcentration, use gel filtration chromatography, collect main peak, the high performance liquid phase purifying.