CN105177032A - Novel human tumor necrosis factor receptor-Fc fusion gene and product protein thereof - Google Patents
Novel human tumor necrosis factor receptor-Fc fusion gene and product protein thereof Download PDFInfo
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Abstract
The invention discloses a method for increasing expression amount of a recombinant human TNFR75-Fc fusion gene. The method comprises the following steps: providing a novel TNFR75-Fc fusion gene of SEQ ID No.:3; further providing a carrier pCI-gs-TNFR75-Fc; in the fusion gene establishment process, introducing 18 alkali groups into a 3'-end primer of TNFR75 and 18 alkali groups into a 5'-end primer of a human IgG1Fc gene fragment for complementation; at the same time improving a cell strain screening method, MSX concentration and a preparation method of gene fragments of human soluble TNFRcDNA and human IgG1 heavy chain constant region Fc fragments in a sequence SEQ ID NO.:3, thereby finally increasing the expression amount of the fusion protein.
Description
The application is application number is 201010268409.3, and the applying date is 2010.09.01, and denomination of invention is the divisional application of new tumor necrosin receptor-Fc fusion gene and product albumen thereof.
Technical field
The invention belongs to genetic engineering pharmaceutical field, be specifically related to a kind of novel TNFR-Fc fusion gene and expression product thereof.
Background technology
Rheumatoid arthritis (RheumatoidArthritis, RA) is one of modal autoimmune disorder of the mankind, and morbidity about 1% is obviously relevant to human leucocyte antigen DR4/DR1, has certain genetic predisposition.The Clinical symptoms of RA mostly is the synovium of joint inflammation of hand, foot, wrist, knee, stern etc., finally causes joint injury.Synovitis causes a large amount of lymphocytic infiltration, mainly comprises scavenger cell, T lymphocyte and plasmocyte, simultaneously blood vessel hyperplasia.Joint injury mainly betides synovial membrane and cartilage and bone adjacent place.
RA is a kind of based on the systemic autoimmune disease involving joint.Recent study shows, tumor necrosis factor-alpha (TNF-α) is the key factor (FeldmannM2001 in RA pathogenesis; RavinderNMaini, 2001; RavinderNMaini, 199), it can stimulate body to produce a series of factor, as the generation of interleukin-(IL)-1, IL-6, IL-8 and granulocyte colony-stimulating factor etc., and inducing endothelial cell expresses adhesion molecule, attract white corpuscle to affected joints.TNF-α also stimulates synovial macrophages, fibroblast, osteoclast and chondrocyte to produce matrix metalloproteinase, and suppresses the synthesis of chondroproteoglycan, and can cause the damage of synovial membrane inflammation and joint cartilage.Block TNF-α and can block inflammatory reaction in the pathogenetic upstream of RA, reach the object for the treatment of RA.
Tumor necrosis factor alpha (TumorNecrosisFactorAlpha, TNF α) and lymphotoxin (Lymphotoxin, LT) play biological action by being combined with its coreceptor TNFR75 and TNFR55.Human tumor necrosis factor receptor 75 (TNFR75) is made up of 461 amino acid, its N-end 235 amino-acid residues, is the cytolemma outskirt fragment that TNFR75 protease hydrolysis gets off, also referred to as soluble TNF R75 (sTNFR75).
Great many of experiments evidence shows that soluble TNF R75 and TNFR55 (i.e. the outer part of the film of acceptor) can block TNF α and LT biological action in vivo (Hunns-MartinLorenz, 2002 by the effective combination with TNF α and LT; Smith, C.A. etc. (1990), science, 248:1019-1023), and TNFR75 distribution is more extensively sent out, affinity is stronger.But the TNFR transformation period is shorter.
The human IgG immunoglobulin like protein transformation period can up to 21 days, and they are most richs in protein in human blood, and be divided into IgA, IgG, IgM, IgE and IgD, wherein IgG has IgG1, IgG2, IgG3 and IgG4 tetra-hypotypes.IgG1Fc section is made up of 232 amino-acid residues, comprises hinge area, CH2 district and CH3 district.Have been reported and the cysteine residues in the Fc hinge region of IgG and other protein (as various cytokine and soluble receptors) are combined and form fusion rotein, make protein-basedly to be similar to IgG molecule but without CH1 region and light chain.Due to structural homology, Fc fusion rotein shows the interior medicine dynamics characteristic suitable with the human IgG of similar isotype.
External existing people constructs the fusion rotein of the Fc fragment of people TNFRII receptor part and IgG by genetic engineering means, this albumen has neutralizing effect (United States Patent (USP) 5,605,590) in vitro to TNF.Domestic also have build this type of fusion rotein, Ma Jing etc. at the recombination of CN1417334A soluble part in tumor necrosis factor acceptor, and construct a kind of fusion rotein containing linker in antigen-4 fusion protein gene and product; The Liu Chang gate of a village etc. constructs a kind of fusion rotein of brachymemma in CN1502632A Novel TNFR-FC fusion protein; Xu Bin etc. utilize bicistronic mRNA to construct a kind of human tumor necrosis factor soluble receptor II-antibody FC section fusion rotein in CN101003575.
This kind of medicine humanization degree is high, and the immunogenic response caused is low, and the treatment works well, than depending merely on anodyne and the more effective symptom management of antiphlogiston.But current this kind of medicine ubiquity preparation method is complicated, the problem that expression amount is low, expensive assistant officer of Denging is to be solved.
Summary of the invention
The problem that the present invention solves there is provided a kind of novel recombinant human TNFR75-Fc fusion gene, and the protein of expressing.
The Second Problem that the present invention solves constructs a kind of recombinant vectors pCI-gs-TNFR75-Fc containing recombinant human TNFR75-Fc fusion gene fragment, after transfection mammalian cell, its eukaryotic amplification label, significantly can improve the copy number of goal gene in host cell, thus improve the expression amount of target protein.
The 3rd problem that the present invention solves is, provides a kind of mammalian cell containing recombinant vectors pCI-gs-TNFR75-Fc, can stably express TNFR75-Fc.Described mammalian cell can be the conventional mammalian cell engineering cell strain such as Chinese hamster ovary celI, NSO cell.
The four problems that the present invention solves is by sulphur ammonia methionine(Met) (MSX) pressurization amplification screening, expression target protein cell strain expression amount is made to improve at least one the order of magnitude, when carrying out highly-expressing cell strain screening, MSX concentration brings up to 400 ~ 500 μm the most at last, and screening mono-clonal expression amount reaches 20 ~ 30pg/cell24hr.
For solving the problem, the present invention implements by following technical solution:
The total serum IgE of extracting HL-60 cell strain, RT-PCR amplification people sTNFR75 gene fragment; Pcr amplification human IgG1 Fc gene fragment.OverlapextentionPCR technology is adopted to carry out two gene fragments to splice the full length cDNA sequence obtaining coding rhTNFR:Fc fusion rotein.This cDNA sequence obtained is inserted clone's intermediate carrier Teasy, confirm through sequencing the structure carrying out carrier for expression of eukaryon correctly.
RhTNFR:Fc fusion rotein cDNA fragment is cloned in expression vector, adopt liposomal transfection teclmiques, the recombinant plasmid rhTNFR:Fc/pCI-gs of aseptic extracting is imported in Chinese hamster ovary celI, through MSX pressurization screening, obtain the gene engineering monoclonal cell strain of the rhTNFR:Fc fusion rotein of high expression level amount, when MSX concentration is brought up to 400 ~ 500 μm, screening mono-clonal expression amount reaches 20 ~ 30pg/cell24hr.
The present invention has synthesized a kind of human tumor necrosis factor receptor fusion gene TNFR75-Fc by the method for OVERLAPPCR, adopt the eukaryotic cell efficient expression vector containing GS system, in mammalian cell, make the expression amount of this fusion rotein improve 20 percentage points through MSX pressurization screening, fusion rotein activity improves 8 percentage points.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but the present invention is not limited to following embodiment.
The preparation of embodiment 1 human soluble TNFR75cDNA
1, pcr amplification TNFR75cDNA
Design primer A:5 ' TC
aAGCTTaTGGCTCCCGTCGCCGTCTGG3 ' primer B:5 ' GTCACAAGATTTGGGCTCGTCGCCAGTGCTCCCTTCAGC3 ',
Design other one group of primer, linker sequence will be introduced in primer B, this sequence (Gly4Ser) 3, be a coding 15 amino acid whose hydrophobic peptides, their mobility is better, do not affect the native three dimensional structure of TNFR and Fc, the DNA sequence dna of its linker is 5 '-GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGC-3 '.
The primer B of linker sequence is introduced in design.Primer Linker-B:5 ' GCTGCCGCCACCGCCGCTTCCGCCACCGCCGCTTCCACCGCCACCGTC.
With people in loop total serum IgE for template, respectively with primer A and B be template, with A and Linker-B for template, carry out RT-PCR reaction, 94 DEG C of sex change 5min start circulation, 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 1min, totally 32 circulations, last 72 DEG C extend 8min.Obtain encode sTNFR75 and the gene fragment contrasting sTNFR75-linker.
2, the qualification of PCR primer and gel reclaim
Above-mentioned condition is carried out PCR, agarose gel electrophoresis is identified, find the band of the about 800bp of target protein molecular weight in product, this fragment is reclaimed.
The clone of embodiment 2 human IgG1 CH Fc fragment gene fragment
Design primer C:5 ' GAGCCCAAATCTTGTGACGAGCCCAAATCTTGTGACAAA3 ', primer D:5 ' AGT
gAATTCtCATTTACCCGGAGACAGGGA3 ';
Design primer linker-C:GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGCGA G.
With IgG1Fc/Teasy vector plasmid for template, respectively with C and D be primer, with Linker-C and D for primer, carry out RT-PCR reaction, 94 DEG C of sex change 5min start circulation, 94 DEG C of 20sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 circulations, last 72 DEG C extend 10min.Amplification obtains the gene fragment of encoding human IgG1Fc and contrast linker-IgG1Fc.
The acquisition of embodiment 3TNFR75-Fc fusion gene
1, the full-length gene fragment of pcr amplification coding rhTNFR-Fc fusion rotein/rhTNFR-linker-Fc
With sTNFR75PCR amplified production and human IgG1 Fc section pcr amplification product for template, with A:5 ' TC
aAGCTTaTGGCTCCCGTCGCCGTCTGG3 ' and D:5 ' AGT
gAATTCtCATTTACCCGGAGACAGGGA3 ' is primer, carries out RT-PCR reaction.
Simultaneously with sTNFR75-linkerPCR amplified production and people linker-IgG1Fc section pcr amplification product for template, with A:5 ' TC
aAGCTTaTGGCTCCCGTCGCCGTCTGG3 ' and D:5 ' AGT
gAATTCtCATTTACCCGGAGACAGGGA3 ' is primer, carries out RT-PCR reaction.
RT-PCR amplified reaction step: 94 DEG C of sex change 5min start circulation, 94 DEG C of 40sec, 62 DEG C of 30sec, 70 DEG C of 1min, totally 30 circulations, last 72 DEG C extend 10min.
Amplification obtains the full-length cDNA fragment of rhTNFR-Fc and rhTNFR-linker-Fc fusion rotein, and its gene fragment is long is respectively 1470bp and 1485bp.
2, rhTNFR-Fc/rhTNFR-linker-Fc fusion rotein cDNA is cloned in intermediate carrier
RhTNFR-Fc/rhTNFR-linker-Fc fusion rotein cDNAPCR amplified production and linear Teasyvector (Promega) carry out ligation.Connect product conversion E.coli E. coli competent host cell, coat add X-gal and IPTG in advance LB flat board (containing 100 μ g/ml penbritins and 15 μ g/ml tsiklomitsins) on carry out blue hickie screening.From transformation plate, the single bacterium colony of random choose white, inoculates propagation respectively, carries out plasmid Rapid identification, obtains TNFR75-Fc and contrast rhTNFR-linker-Fc fusion gene.
The expression of embodiment 4TNFR75-Fc/rhTNFR-linker-Fc fusion gene
1, rhTNFR-Fc/rhTNFR-linker-Fc fusion gene fragment is cloned in expression vector
The structure of rhTNFR-Fc/rhTNFR-linker-Fc expressing fusion protein type recombinant plasmid is the EcoRI single endonuclease digestion site utilizing the pCI-gs transformed through business expression vector pCI-neo, insert through the correct rhTNFR-Fc/rhTNFR-linker-Fc gene fragment of determined dna sequence result, with the both forward and reverse directions of Spe1 single endonuclease digestion qualification Insert Fragment, connect product conversion E.coli competent cell, after Screening and Identification, obtain rhTNFR-Fc/pCI-gs/E.coliTop10F ' and contrast rhTNFR-linker-Fc/pCI-gs/E.coliTop10F ' recombinant bacterium.
2, a large amount of preparations of expression type recombinant plasmid
The strain Escherichia coli carrying recombinant plasmid rhTNFR-Fc/pCI-gs/rhTNFR-linker-Fc/pCI-gs is inoculated in 200ml liquid nutrient medium, after incubated overnight, adopt QiagenPlasmidMaxiKits, carry out a large amount of preparations of recombinant plasmid to specifications.Gained plasmid DNA, through 70% washing with alcohol, aseptic air-dry after, be dissolved in 500 μ l sterilized waters, get 2 μ l plasmids and dilute 100 times and detect A260 value and A280 values respectively, when the ratio of A260/A280 is between 1.75-1.85, then this plasmid is used for cell transfecting.
3, cell strain transfection
The Chinese hamster ovary celI cultivating two days in culturing bottle, with after 0.25% trypsin solution digestion, is got and is carried out Trypan Blue counting in right amount, with 3 × 10
5individual cell count inoculates the single hole of 6 orifice plates; Cell is inoculated 6 orifice plates and is reached 90% full layer after 24 hours, can carry out transfection; Respectively get 250 μ lOPTI-MEMI (Gibco), add in two sterile centrifugation tube, in two pipes, add 10 μ lLipofectamine (Invitrogen) and 4 μ g respectively dilute plasmid, by in the liquid mixing in two pipes to same pipe, room temperature leaves standstill and makes it form DNA in 20 minutes: liposome complex; Meanwhile, inhale the cell culture medium abandoned in 6 orifice plates, in each hole, add 2ml cell growth medium with after cell growth medium (DMEM/F12/10%FCS) gently washed cell twice; By 500 μ lDNA: liposome complex solution adds in the respective aperture in 6 orifice plates, be placed in 37 DEG C, 5%CO
2overnight incubation in incubator.
The screening of embodiment 5TNFR75-Fc/TNFR75-linker-Fc fusion rotein overexpression cell line
Embodiment 4 is screened obtained recombinant cell strain with after 0.25% trypsin solution digestion, import overnight incubation in three 100mm culture dish into the ratio of 1:3; After 24 hours, substratum in 100mm culture dish is replaced by Selective agar medium, namely in DMEM (without the glutamine) nutrient solution containing 10% dialysis foetal calf serum (GibcoBRL), adding final concentration is that the MSX (thiamines methionine(Met), Sigma) of 25 μm is as selective agent; Selective agar medium was changed every 2-4 days according to necrocytosis situation, an about 7-10 days cell starts mortality, after about 14 days, cell clone starts to occur, transfer in 24 orifice plates with clone's ring from picking mono-clonal flat board when cell clone diameter reaches 1-2mm, often write instructions and transfer dye picking about 50 single cell clones.
When single cell clone grows to the full layer of 50-70% in 24 orifice plates, the culture supernatant of getting each clone carries out ELISA detection, according to cell growth status and expression amount, chooses the cell clone that expression amount is greater than 1pg/cell24hr and carries out medicine pressurization amplification screening; The concentration progressively improving selective agent MSX makes the recombinant plasmid be integrated in karyomit(e) increase, and when drug level is increased to 500 μm, screening mono-clonal expression amount reaches 20 ~ 30pg/cell24hr.Get the culture supernatant of each clone, application ELISA method detects its fusion protein expression, and each picking mono-clonal 50 carries out expression amount mensuration altogether, the results are shown in Table 1.
Table 1, expression amount contrast and experiment
Calculated from table 1, produce novel restructuring TNFR75-Fc expression amount by the present invention and improve 20 percentage points than control experiment, be more conducive to the expanding production of this albumen.
The active simultaneous test of embodiment 6TNFR75-Fc/TNFR75-linker-Fc fusion rotein
The fusion rotein TNFR75-Fc/TNFR75-linker-Fc expressed with the cell strain filtered out is carried out Sepharose-A affinity chromatography and hydrophobic chromatography.Concrete steps be (1) by protein A affinity chromatography purifying protein, in protein A affinity chromatography, during albumen loading, add the urea of 1mol/L, in the elution process of albumen, in elutriant, also add the urea of 1mol/L urea.Target protein under wash-out low pH about 3.5 time; (2) (1) gained sample is directly passed through hydrophobic chromatography loading, salts solution used is ammonium sulfate, and filler used is the filler containing butyl or phenyl.Obtain the fusion rotein that purity is greater than 90%.
Then active determination test is carried out.By rhTNFR-Fc anti-tnf-alpha short of money the biologic activity detecting rhTNFR-Fc is killed and wounded to target cell L929 cell strain.
Sample activity (AU/ml)=[standard substance ED
50(ng/ml)/product to be tested ED
50(ng/ml)] × standard substance are tired (AU/ml) × testing sample extension rate
The results are shown in Table 2.
The L929 cell of 1, taking the logarithm vegetative period, with cell dispersion abundant after 0.25% trypsinase conventional digestion, surveys live (1 piece of 96 orifice plate) at every turn and gets about 2 × 10
6cell, centrifugally abandons supernatant, adds nutrient solution B and the cell density of cell culture fluid is adjusted to 1.5 × 10
5after/ml, add in 96 well culture plates, 0.1ml/ hole, be placed in 37 DEG C, 5% carbonic acid gas incubator overnight incubation (18 ~ 24h).
2, prepare standard substance: the TNFR:Fc working standard getting 10 μ g/ml, it is 500ng/ml that the dactinomycin solution adding the 10 μ g/ml diluted with TNF-α (40U/ml) is diluted to concentration.
3, according to the protein quantification of sample to be determined, first 1mg/ml is diluted to sterile water for injection, the dactinomycin solution of the 10 μ g/ml diluted with TNF-α (40U/ml) in 1.5ml sterile centrifugation tube is progressively diluted to 500ng/ml, notices that each extension rate is no more than 10 times.
4, the dilution of standard substance and trial-product
The diluent of continuous doubling dilution working standard or sample (namely contain TNF-α and the actinomycetes D of same concentrations after dilution in all pipes, but the concentration of TNFR:Fc is different.When next step adds in the hole containing equal-volume nutrient solution, actual extent of dilution will be now 2 times).
5, every hole adds the various dilution sample of 100 μ l, each extent of dilution 3 holes.Negative control adds the dactinomycin solution of the 40U/ml with TNF-α dilution.The dactinomycin solution of the 10 μ g/ml with the dilution of serum-free RPMI-1640/DMEM (1:1) nutrient solution is added in positive control.
6,37 degree of cultivation 18-24 hour in 5%CO2 incubator.
7, endpoint determination: every hole adds 40.0 μ l violet staining liquid, dyes ten minutes, with tap water rinse 3 ~ 4 times, until tap water is colourless.Patted dry by 96 orifice plates as far as possible, make do not have residual moisture in plate, add destainer to 96 orifice plates, every hole 100 μ l, mixing, microplate reader 570nm place reads absorption value.The results are shown in Table 2.
Table 2, active contrast and experiment
| LOGEC50 | |
| TNFR-Fc | 3.812 |
| TNFR-linker-Fc | 3.518 |
Calculated from table 2, under same protein concentration, TNFR75-Fc improves 8 percentage points than the protein-active of TNFR75-linker-Fc.
Claims (2)
1. improve a method for TNFR75-Fc track fusion amount, it is characterized in that, comprise the steps: to build a kind of restructuring TNFR75-Fc fusion gene containing base sequence described in SEQIDNO:3; Build a kind of recombinant vectors containing the base sequence described in SEQIDNO:3, described carrier is pCI-gs-TNFR75-Fc, and this carrier is carrying out in highly-expressing cell strain screening process, and MSX concentration brings up to 500 μm the most at last; PCI-gs is transformed by pCI-neo and obtains, and utilizes the EcoRI single endonuclease digestion site of pCI-gs, inserts through the correct rhTNFR-Fc gene fragment of determined dna sequence result, with the both forward and reverse directions of Spe1 single endonuclease digestion qualification Insert Fragment, obtains pCI-gs-TNFR75-Fc.
2. method as claimed in claim 1, is characterized in that, in described SEQIDNO:3 Fusion gene construction process, introduces 18 base complementrities that 18 bases and human IgG1 Fc gene fragment 5 ' are held in 3 ' the end primer of TNFR75;
In described SEQIDNO:3 sequence, the preparation method of human soluble TNFR75cDNA is:
1), pcr amplification TNFR75cDNA
Design primer A:5 ' TCAAGCTTATGGCTCCCGTCGCCGTCTGG3 ' primer B:5 ' GTCACAAGATTTGGGCTCGTCGCCAGTGCTCCCTTCAGC3 ',
Design other one group of primer, linker sequence will be introduced in primer B, this sequence (Gly4Ser) 3, be a coding 15 amino acid whose hydrophobic peptides, their mobility is better, do not affect the native three dimensional structure of TNFR and Fc, the DNA sequence dna of its linker is 5 '-GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGC-3 ';
The primer B of linker sequence is introduced in design; Primer Linker-B:5 ' GCTGCCGCCACCGCCGCTTCCGCCACCGCCGCTTCCACCGCCACCGTC;
With people in loop total serum IgE for template, respectively with primer A and B be template, with A and Linker-B for template, carry out RT-PCR reaction, 94 DEG C of sex change 5min start circulation, 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 1min, totally 32 circulations, last 72 DEG C extend 8min; Obtain encode sTNFR75 and the gene fragment contrasting sTNFR75-linker;
2), the qualification of PCR primer and gel reclaim
Above-mentioned condition is carried out PCR, agarose gel electrophoresis is identified, find the band of the about 800bp of target protein molecular weight in product, this fragment is reclaimed;
The clone of human IgG1's CH Fc fragment gene fragment in described SEQIDNO:3 sequence:
Design primer C:5 ' GAGCCCAAATCTTGTGACGAGCCCAAATCTTGTGACAAA3 ', primer D:5 ' AGTGAATTCTCATTTACCCGGAGACAGGGA3 ';
Design primer linker-C:GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGCGA G;
With IgG1Fc/Teasy vector plasmid for template, respectively with C and D be primer, with Linker-C and D for primer, carry out RT-PCR reaction, 94 DEG C of sex change 5min start circulation, 94 DEG C of 20sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 circulations, last 72 DEG C extend 10min; Amplification obtains the gene fragment of encoding human IgG1Fc and contrast linker-IgG1Fc.
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| CN108355141B (en) * | 2018-01-11 | 2019-06-18 | 广西壮族自治区人民医院 | A kind of drug and preparation method thereof of the treatment allergic disease based on TIM-4-Fc fusion protein |
| CN112494658A (en) * | 2020-12-04 | 2021-03-16 | 苏桥生物(苏州)有限公司 | Stable Fc fusion protein preparation |
| WO2023044774A1 (en) * | 2021-09-24 | 2023-03-30 | Sichuan Clover Biopharmaceuticals, Inc. | Tpo mimetic fusion proteins and methods of use submission of sequence listing as ascii text file |
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| US5605690A (en) * | 1989-09-05 | 1997-02-25 | Immunex Corporation | Methods of lowering active TNF-α levels in mammals using tumor necrosis factor receptor |
| WO1997037016A1 (en) * | 1996-04-02 | 1997-10-09 | Yeda Research And Development Co. Ltd. | Modulators of tnf receptor associated factor (traf), their preparation and use |
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| CN105177032B (en) | 2019-05-10 |
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