CN1244696C - Highly effective expressing method of interleukin-12 - Google Patents
Highly effective expressing method of interleukin-12 Download PDFInfo
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- CN1244696C CN1244696C CN 03131567 CN03131567A CN1244696C CN 1244696 C CN1244696 C CN 1244696C CN 03131567 CN03131567 CN 03131567 CN 03131567 A CN03131567 A CN 03131567A CN 1244696 C CN1244696 C CN 1244696C
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Abstract
The present invention relates to a high-efficiency expression method of interleukin-12, particularly to a method for preparing interleukin-12(IL-12) by biological technology. In the method, P40 subunit coding region cDNA of IL-12 is inserted into a pcDNA3 eukaryotic expression carrier, P35 subunit coding region cDNA of IL-12 is inserted into a pEE14 eukaryotic high-efficiency expression carrier, and two kinds of eukaryotic cell recombinant plasmid of pcDNA3/p40 and pEE14/p35 are respectively constructed. After host cells are cotransfected, G418 and MSX are simultaneously added into a sieving culture medium to be sieved to select survival positive clone which is pressurized and enlarged so as to obtain engineering cell strains of high-expression IL-12. The method is simple and convenient to utilize the characteristics of natural unbalanced expression of P40 and P35 of IL-12, and adopts two carriers having different expression efficiencies so as to balance the expression of two chains.
Description
One, technical field
The present invention relates to a kind ofly prepare the method for interleukin 12 (IL-12) by biotechnology, the expression method of particularly a kind of IL-12 that recombinates belongs to technical field of bioengineering.
Two, background technology
IL-12 can promote the propagation and the killing activity of NK cell, T cell, induces production of cytokines such as gamma-interferon, regulates the Th1 cells whose development, has good potential applicability in clinical practice.Entered at present the clinical experiment stage of antitumor, antiviral property disease and treatment asthma.Natural IL-12 is a kind of heterodimer cytokine that is mainly produced by scavenger cell and B cell, is made up of P40 and two subunits of P35.The generation of P40 is much larger than P35 in the cell cytosol, but has only P40 through the heterodimer that disulfide linkage is connected biologic activity to be arranged just with P35.
Some researchists insert the gene of P40 and P35 respectively respectively and carry out cotransfection with a kind of carrier and (wear swallow before the present invention, Chen Weifeng. the expression of people's recombinant interleukin 12 in Chinese hamster ovary celI. biological chemistry and biophysics progress, 1998,25 (3): 254-258), or in single plasmid, make up the carrier (Liu Meng contain P40 and P35 Expression element respectively, Zheng Shanshan. recombinant human cytokine 12 genes are at the stably express of CHO-dhfr-cell. Journal of Immunology, 1996,12 (4): 215-219), also there is the people that the gene fusion structure IL-12 strand carrier for expression of eukaryon of P40 and P35 is expressed (Zhang Mei, department carries out and gives birth to, Jin Li etc. the structure of recombined human single-chain interleukin 12 fusion genes, eukaryotic expression and Determination of biological activity. Chinese Journal of Hematology, 2000,21 (12): 636-640), these methods all fail to realize efficiently expressing of IL-12.The gene of P40 and P35 inserted respectively with a kind of carrier carry out cotransfection, because the efficient of cotransfection is lower, this method causes very big difficulty for the colony screening of expressing IL-12, is difficult to obtain high-expression clone, and the expression of P40 and P35 is seriously uneven.In single plasmid, make up the carrier that contains P40 and P35 Expression element respectively, how accurate this method no matter carry out balance, because the order difference of two goal gene on carrier, usually hinder transcribing of goal gene that the downstream promotor carries when the goal gene that upstream promoter carries is transcribed, the expression of P40 and P35 is also uneven.Making up IL-12 strand carrier for expression of eukaryon expresses, though this kind method can make the expression balance of two chains, but owing to understand very few to the space structure of IL-12, the adding of joint might influence the folding and space structure of two chains of IL-12, thereby influence its biologic activity, the biologic activity report of the IL-12 that this kind method is expressed differs.
Three, summary of the invention
1, technical problem
The purpose of this invention is to provide a kind of easy P40 that utilizes IL-12 and the expression method of the unbalanced characteristic of the natural expression of P35, two kinds of different carriers of expression efficiency of employing equilibrated interleukin 12 so that the expression of two chains is tried one's best.
2, technical scheme
The coding region cDNA of P40 subunit with IL-12 inserts the pcDNA3 carrier for expression of eukaryon respectively, the coding region cDNA of P35 subunit of IL-12 is inserted pEE14 eucaryon efficient expression vector, made up the different eukaryotic cell recombinant plasmid of two kinds of expression efficiencies of pcDNA3/p40, pEE14/p35 respectively, behind the cotransfection host cell, in screening culture medium, add G418 (Geneticin) simultaneously and methionine sulfoxide (MSX) screens, the positive colony of picking survival is identified, obtains the positive colony of high expression level IL-12; Be further to improve the expression amount of IL-12, with MSX to the amplification of pressurizeing of the positive colony of each high expression level IL-12, the engineering cell strain that acquisition has industrialization value.Perhaps, to the amplification of pressurizeing of the positive colony of each high expression level IL-12, obtain engineering cell strain with the combination of G418 and MSX with industrialization value.
Wherein, the gene of IL-12 comprises the cDNA of people or Mammals IL-12.P40 and the coding region cDNA of P35 subunit can directly buy on market, also can adopt the amplification of polymerase chain reaction (PCR) technology to obtain.Host cell can adopt Chinese hamster ovary cell (Chinese hamster ovary celI) or Non-secreting cell mammalian cells such as (NS0 cells).The combination of used G418 and MSX can be the cooperation between both any concentration during the pressurization amplification.
3, beneficial effect
Adopt two kinds of carrier cotransfection host cells that expression efficiency is different; Combination with G418 and MSX is increased to each positive colony pressurization, and these are the distinctive distinguishing characteristicss of the present invention.Compared with the prior art, advantage of the present invention is to utilize P40 and two unbalanced characteristics of the natural expression of chain of P35 of IL-12, they are inserted two kinds of carriers that expression efficiency is different respectively, be easy to high expression level under the P40 subunit native state, the coding region cDNA of P40 subunit is inserted the lower pcDNA3 carrier for expression of eukaryon of expression efficiency, expression amount is lower under the P35 subunit native state, the coding region cDNA of P35 subunit is inserted the higher pEE14 eucaryon efficient expression vector of expression efficiency, the balance so that the expression of two chains is tried one's best.Do not change simultaneously the structure of IL-12, do not influence the biologic activity of IL-12.The screening sign difference of two kinds of carriers helps filtering out the positive colony of expressing two chains of IL-12 simultaneously during the recombinant vectors cotransfection host cell.And the gene amplification system that contains in the utilization carrier to each positive colony pressurization amplification, can further improve expression amount.Especially adopt the combination of G418 and the MSX amplification of pressurizeing, arbitrarily equal effective than independent use MSX of the combination of concentration.
Four, description of drawings
Fig. 1 is the electrophoretogram of the PCR product of people IL-12p40cDNA, and wherein swimming lane 1 is the pcr amplification product of p40, and swimming lane 2 is λ DNA/HindIII+EcoRI Marker;
Fig. 2 is a pcDNA3/p40 construction of recombinant plasmid schema;
Fig. 3 is the electrophoretogram of the PCR product of people IL-12p35cDNA, and wherein swimming lane 1 is the pcr amplification product of p35, and swimming lane 2 is λ DNA/HindIII+EcoRI Marker;
Fig. 4 is a pEE14/p35 construction of recombinant plasmid schema;
Fig. 5 is that pcDNA3/p40, pEE14/p35 stablize cotransfection CHO-K1 cell synoptic diagram;
Fig. 6 is the Western Blot result behind the polyacrylamide gel electrophoresis under the non-reductive condition, and swimming lane 1 is molecular weight of albumen standard (85.9kD, 68.8kD, 52.5kD, 40.0kD, 28.4kD), swimming lane 2 is empty plasmid transfection contrast, the culture supernatant after the positive clone's pressurization of swimming lane 3-5;
Fig. 7 is the Western Blot result behind the polyacrylamide gel electrophoresis under the reductive condition, swimming lane 1 is molecular weight of albumen standard (85.9kD, 68.8kD, 52.5kD, 40.0kD 28.4kD), swimming lane 2 is empty plasmid transfection contrast, swimming lane 3 is IL-12 standard (a peproTech company), the culture supernatant after the positive clone's pressurization of swimming lane 4-5;
Fig. 8 is that the transfection CHO cell culture supernatant is to the hormesis of PHA activatory PBMC propagation and the comparison of standard substance.
Five, embodiment
1.1 pcDNA3/p40 construction of recombinant plasmid
With plasmid T carrier/p40 of extracting is the p40 gene that template is carried out pcr amplification people IL-12, estimates length 1003bp (as Fig. 1), and parameter is: 94 ℃ 1 minute, 62 ℃ 1 minute, 72 ℃ 2 minutes, circulate last prolongation 7 minutes 28 times.PcDNA3/p40 construction of recombinant plasmid flow process (see figure 2): behind pcDNA3 plasmid usefulness HindIII and BamHI double digestion recovery purifying, be connected product transformed competence colibacillus bacillus coli DH 5 alpha, screening positive recombinant with the PCR product of same double digestion.Positive recombinant plasmid is entrusted the order-checking of Dalian precious biotinylated biomolecule technology company limited, and the result shows that the p40cDNA sequence is consistent with report.
1.2 pEE14/p35 construction of recombinant plasmid
With plasmid T carrier/p35 of extracting is the p35 gene that template is carried out pcr amplification people IL-12, estimates length 676bp (as Fig. 3), and parameter is: 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute, circulate last prolongation 7 minutes 30 times.PEE14/p35 construction of recombinant plasmid flow process (see figure 4): after the pEE14 plasmid that the SmaI enzyme is cut reclaims purifying; carrying out flush end with PCR product that the Klenow enzyme is mended flat p35 goal gene is connected; product transformed competence colibacillus bacillus coli DH 5 alpha, the screening positive recombinant.Positive recombinant plasmid is entrusted the order-checking of Dalian precious biotinylated biomolecule technology company limited, and the result shows that the p35cDNA sequence is consistent with report.
1.3 pcDNA3/p40, pEE14/p35 stablize cotransfection CHO-K1 cell with liposome method
The heterodimer molecule that complete IL-12 molecule is made up of two different subunits is only expressed these two subunits simultaneously in same system, and makes their correct assemblings, the IL-12 that just might obtain to have biologic activity.PcDNA3/p40, pEE14/p35 cotransfection CHO-K1 cell and positive-selecting (see figure 5): the CHO-K1 cell cultures is in containing the F-12 substratum of 10% calf serum, by every hole 3 * 10
5The amount of cell is inoculated the CHO-K1 cell of exponential phase of growth, 37 ℃ of 5%CO in six orifice plates
2Be cultured to the about 80% remittance sheet of cell.PcDNA3/p40, pEE14/p35 recombinant plasmid extract in a large number, behind the polyoxyethylene glycol method purifying, get each 1 μ g of pcDNA3/p40, pEE14/p35, mix.With LIPOFECTAMINETM Reagent transfection CHO-K1 cell, 37 ℃ of 5%CO
2Cultivate after 72 hours, 0.25% tryptic digestion imports screening DMEM substratum (containing final concentration is the MSX of 25 μ M and the G418 of 600mg/L) into by dilution in 1: 4.After cultivating about 14 days, five resistance clones appear.Picking the clone be transferred in 24 orifice plates, transfers in six orifice plates and cultivate, and 0.25% tryptic digestion extracts RNA, carries out the expression that RT-PCR identifies p40mRNA, p35mRNA, gets culture supernatant simultaneously and carries out ELISA and Determination of biological activity.5 resistance clones that obtain all have IL-12 to express.Choose 1 higher strain clone of expression IL-12 and carry out MSX pressurization amplification, MSX establishes 100,250,500 μ M isoconcentration gradients, and acquisition has the engineering cell strain (seeing following table for details) of the high expression level IL-12 of industrialization value.
The amplification of foreign gene in mammalian cell is to improve foreign gene to express one of important method of output in mammalian cell.Because the generation of natural P40 much larger than P35, adopts glutamine synthetase (GS) gene of pEE14 to make the gene of P35 obtain the high-level efficiency amplification as dominant gene amplification selective marker in the present embodiment, with as far as possible with the expression balance of P40.From embodiment 1, choose and express 1 higher strain clone of the IL-12 amplification of pressurizeing, MSX establishes 100,250,500 μ M isoconcentration gradients, G418 establishes 600,800,1000,1200mg/L isoconcentration gradient, MSX and each concentration gradient of G418 are carried out various combination, add substratum and carry out the pressurization amplification of goal gene, cultivated nutrient solution of middle replacing 10-14 days.The clone of picking survival gets its culture supernatant Western Blot and analyzes Recombinant Protein Expression and measure expression amount with elisa technique, and acquisition has the engineering cell strain of the high expression level IL-12 of industrialization value.
Positive colony is IL-12 expression amount (ng/ml) after MSX or MSX and G418 different concns gradient combination pressurization amplification:
| ?MSX?100 | ?MSX?250 | ?MSX?500 | |
| ?G418?0 | ?410 | ?500 | ?725 |
| ?G418?600 | ?595 | ?635 | ?735 |
| ?G418?800 | ?460 | ?840 | ?912 |
| ?6418?1000 | ?568 | ?845 | ?994 |
| ?G418?1200 | ?555 | ?565 | ?775 |
The qualification process of positive colony:
1. the IL-12 expression amount in enzyme-linked immunosorbent assay (ELISA) the technical measurement culture supernatant is pcDNA3/p40, pEE14/p35 stablize cotransfection CHO-K1 cell with liposome method after in positive colony screening and the follow-up pressurization amplification procedure, all adopt people IL-12 ELISA test kit (peproTech company) as the rapid screening means, get the secretory volume of culture supernatant detection IL-12, schedule of operation is undertaken by the test kit specification sheets.
2. mRNA transcription analysis
After amplification cultivation, the guanidinium isothiocyanate method is extracted RNA and is carried out RT-PCR and analyze, and in 5 strain resistance clones, transcribing of p35mRNA and p40mRNA is arranged all to the resistance clone that obtains.
3. immunoblotting (Western Blot) is analyzed the expression of IL-12
PEE14 empty carrier transfection clone's culture supernatant compares, to the higher clone of expression amount who obtains, cultivate after 7 days, Western blot analyzes after getting the culture supernatant polyacrylamide gel electrophoresis, positive band (see figure 6) appears in non-reduced electrophoresis at the 70kD place, the reduction electrophoresis band of expression (see figure 7) occurs at 40kD and 35kD place.
4. the activation analysis of IL-12
Get people's fresh anticoagulant heparin peripheral blood, the dilution of equal-volume Hanks liquid.Separate mononuclearcell (PBMC) with Ficoll.After Hanks liquid is washed twice, be suspended from the RMPI1640 perfect medium that contains 10% calf serum, adjusting cell concn is 1 * 10
6Individual/ml, add phytohaemagglutinin (PHA) (final concentration is 10 μ g/ml), by 100 μ l/ holes cell is added 96 orifice plates.Cultivate after 72 hours, add different dilution IL-12 standard substance and supernatant to be measured again.Cultivate after 48 hours, every hole adds 10 μ l tetramethyl-azo azoles salt (MTT), continues to cultivate about 4-6 hour, and is centrifugal, removes 100 μ l supernatants.Every hole adds the 10mmol/LHCL that 100 μ l contain 10%SDS, allows reduzate fully dissolve.Put on the enzyme connection detector and measure the OD value, detect wavelength 570nm, reference wavelength 630nm.To the mapping of diluted sample degree, standard of comparison product curve and testing sample curve can they record the content of cytokine in the testing sample with the OD value.Culture supernatant is done dilution in 1: 2500,1: 5000,1: 10000,1: 20000,1: 40000 successively, measure proliferation activity result such as Fig. 8 that it promotes PHA activated PBMC.Find still can to promote the human PBMC to rise in value after 40000 times of the transfection CHO cell culture supernatant dilutions, extrapolate expression amount with standard substance after relatively and be about 1000ng/ml.
Transfection the dynamic observing and stability analysis of Chinese hamster ovary celI strain secretion IL-12 of IL-12 cDNA
We secrete dynamically observing of IL-12 to the engineering cell strain that obtains, this cell strain remove went down to posterity 6 months under the MSX pressure condition after, still can stably excreting IL-12, show that the Chinese hamster ovary celI strain that can efficiently express IL-12 that obtains through screening is more stable, have industrialization value.
Claims (2)
1, a kind of expression method of interleukin 12, it is characterized in that respectively the coding region cDNA of P40 subunit with IL-12 inserts the pcDNA3 carrier for expression of eukaryon, the coding region cDNA of P35 subunit of IL-12 is inserted the pEE14 carrier for expression of eukaryon, make up the different eukaryotic cell recombinant plasmid of two kinds of expression efficiencies of pcDNA3/p40, pEE14/p35 respectively, behind the cotransfection host cell, in screening culture medium, add G418 simultaneously and methionine sulfoxide screens, the positive colony of picking survival is identified, obtains the positive colony of the IL-12 of high expression level; To the positive colony of the expressing IL-12 amplification of pressurizeing, acquisition has the engineering cell strain of the high expression level IL-12 of industrialization value with methionine sulfoxide.
2, the high-efficiency expression method of interleukin 12 according to claim 1 is characterized in that with the combination of G418 and methionine sulfoxide the amplification of pressurizeing of the positive colony of high expression level IL-12 is obtained the engineering cell strain of IL-12.
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| CN102268407B (en) * | 2011-07-20 | 2014-04-02 | 中国科学技术大学 | Large-scale serum-free culture method for rhIL-12 engineering cells |
| CN103194480A (en) * | 2012-01-06 | 2013-07-10 | 中国科学技术大学 | High-efficiency expression method of human interleukin-10 (hIL-10) |
| CN103360461B (en) * | 2012-03-31 | 2016-12-14 | 中国科学技术大学 | A kind of people's IL-12 affinity purification chromatographic column, its preparation method and the method utilizing its purification of recombinant human IL-12 |
| CN102876684B (en) * | 2012-09-17 | 2014-10-08 | 青岛康立泰药业有限公司 | Coding gene, eukaryotic host cell and expression method of human interleukin-12 |
| CN110964695B (en) * | 2019-12-10 | 2023-05-30 | 康立泰生物医药(青岛)有限公司 | Cell strain and method for detecting rhIL-12 in-vitro activity by proliferation of cell strain |
| CN112442504B (en) * | 2020-12-10 | 2023-01-24 | 电子科技大学 | Preparation method of recombinant grass carp interleukin-12 active protein |
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