Three-cistron expression vector, preparation method and application

An expression vector, tricistron technology, applied in the field of genetic engineering, can solve the problems affecting the quality of monoclonal antibodies, the problems of industrialized production of genetic engineering, positive clones not expressing the target protein, etc., so as to improve the expression level of antibody protein and improve the Clonal selection rate, the effect of improving effectiveness

Active Publication Date: 2016-06-22
河南普诺易生物制品研究院有限公司 +1
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Problems solved by technology

However, the defect of this type of vector is that the ratio of heavy chain to light chain expression is unbalanced, and the ratio of the two affects the quality of monoclonal antibodies, such as aggregation and glycosylation (IRES-mediatedTricistronic vectors for enhancing generation of high monoclonal antibody expressing CHOcelllines.JBiotechnol.2012)
Although some vectors can express antibody heavy chain and light chain at the same time (patent application number: 201110380100.8, a high-efficiency expression vector of antibody and its preparation method), but because the selection marker gene is not in the same coding frame as the heavy chain and light chain genes, Some positive clones screened will not express the target protein, and false positive ce

Method used

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  • Three-cistron expression vector, preparation method and application
  • Three-cistron expression vector, preparation method and application
  • Three-cistron expression vector, preparation method and application

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Embodiment 1

[0038] The construction of tricistronic expression vector comprises the following steps:

[0039] 1. Construction of pIRES-101 vector (i.e., on the basis of pIRES-Neo vector, replace CMV promoter with SV40 promoter, insert light chain signal peptide sequence, and NdeI, HpaI restriction sites in MCS at the same time)

[0040] 1) Synthesize the fusion sequence of SV40 promoter and light chain signal peptide

[0041] According to the reported SV40 promoter sequence (GenBank accession number: KM359772.1, bases 2794-3247, as shown in SEQ ID NO: 3) and light chain signal peptide sequence (GenBank accession number: Z69026.1, bases 1-66 base, as shown in SEQ ID NO: 4) to artificially synthesize the fusion sequence of SV40 promoter and light chain signal peptide (as shown in SEQ ID NO: 9), which was specifically synthesized by General Biogene (Anhui) Co., Ltd. To facilitate cloning, when synthesizing the fusion sequence, AGCACGCGT sequence was introduced at the 5' end, where AGC was t...

Embodiment 2

[0092] The construction of the pIRES-106 vector comprises the following steps:

[0093] 1) Artificially synthesized anti-CD20 antibody kappa chain cDNA sequence and constructed pIRES-105C expression vector

[0094] The anti-CD20 antibody kappa chain cDNA sequence (as shown in SEQ ID NO: 14) was artificially synthesized according to the sequence shown in SEQ ID NO: 8, and was synthesized by General Biogene (Anhui) Co., Ltd. For the convenience of cloning, ATACATATG was introduced at the 5′ end of the synthetic sequence, where ATA was the protective base and CATATG was the NdeI restriction site; GTTAACAGC was introduced at the 3′ end, where GTTAAC was the HpaI restriction site and AGC was the protective base. Then the synthetic anti-CD20 antibody kappa chain cDNA sequence was inserted into the light chain expression cassette of pIRES-105.

[0095] The cDNA sequence of the anti-CD20 antibody kappa chain and pIRES-105 plasmid DNA digested with NdeI / HpaI double enzyme digestion sy...

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Abstract

The invention discloses a three-cistron expression vector, a preparation method and application and belongs to the technical field of gene engineering.The three-cistron expression vector comprises a nuclear matrix combining region sequence and a three-cistron sequence which is formed in the mode that two internal ribosomes enter a site sequence and connected; the structure of the three-cistron sequence is promoter-light chain signal peptide SPL-IRESwt-heavy chain signal peptide SPH-IRESatt-selection marker-Poly A.The vector can express a light chain, a heavy chain and a selection marker gene of an antibody at the same time, the problem that expression of a heavy chain, a light chain and a selection marker gene of a traditional vector is not balanced is solved, antibody quality is improved, and the cloned screening rate of positive cells is raised; meanwhile the nuclear matrix combining region sequence contained on the vector can further overcome transgene silencing, efficient and long-term expression of transgenes in host cells is achieved, on one hand, the antibody protein expression level is improved, and on the other hand, efficiency of screening of subsequent monoclonal antibody cell lines is improved.

Description

technical field [0001] The invention relates to a tricistronic expression carrier, and also relates to a preparation method and application of the expression carrier, belonging to the technical field of genetic engineering. Background technique [0002] Antibody is a kind of immunoglobulin with special amino acid sequence induced by antigen, synthesized and secreted by lymphocytes (plasma cells). Monoclonal antibodies (Monoclonal antibodies, mAbs) are highly specific, and thus are widely used in the treatment of various diseases, especially cancer and autoimmune diseases. Since the first monoclonal antibody drug Orthoclone Okt3 was launched in 1986, 35 monoclonal antibody drugs have been approved for marketing. At present, monoclonal antibodies are the fastest-growing class of biomedical molecules, and have attracted more and more attention from scholars and pharmaceutical companies. Most monoclonal antibodies (IgG) are produced from two identical heavy chain (HC) and two ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/65C12N15/67
CPCC12N15/63C12N15/65C12N15/66C12N15/67C12N2840/203
Inventor 王天云贾岩龙倪天军赵春澎徐红彦王喜成陈思佳郭潇
Owner 河南普诺易生物制品研究院有限公司
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