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A mammalian cell expression vector for industrial production

An expression vector and mammalian technology, applied in the field of molecular biology and biomedicine, can solve problems such as expensive, difficult to handle in the optimization process, and failure to screen high-expression cell lines

Active Publication Date: 2022-03-22
济南海湾生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a result, many low-yielding clones will survive the initial selection process, making optimization very intractable and expensive, and failing to select high-expressing cell lines

Method used

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  • A mammalian cell expression vector for industrial production
  • A mammalian cell expression vector for industrial production
  • A mammalian cell expression vector for industrial production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of the vector

[0041] The sequences of the CMV promoter and its TATA box, mouse DHFR, BGH polyA (BGHpA), beta-globin polyA (bGpA), and TK polyA (TKpA) were obtained from GenBank, and then GAATTC (EcoRI digestion) in order from 5' to 3' site)-CMV promoter-Kozak sequence-BamHI and SalI restriction sites-BGHpA-SynpA-bGpA-CMV TATA box-DHFR-TKpA-GAATTC (XhoI restriction site) after sorting out the sequence, by artificial synthesis such as The DNA fragment shown in SEQ No. 5; EcoRI and XhoI digested and inserted into the pUC19 plasmid, the plasmid successfully inserted into the above DNA fragment after inspection is the vector used for protein expression in mammalian cells.

Embodiment 2

[0042] Example 2 Expression of GFP (green fluorescent protein).

[0043] 2.1 Vector construction and expression

[0044] Four different vectors were constructed with GFP as the target protein: a) pCMV- GFP- SV40- DHFR- TKpA, b) pCMV- GFP- BGHpA- SynpA- bGpA- SV40- DHFR- TKpA, c) pCMV- GFP- BGHpA- TATA-DHFR-TKpA and d) pCMV-GFP-BGHpA-SynpA-bGpA-TATA-DHFR-TKpA, the steps are as follows: refer to GenBank to obtain the sequences of GFP (LN515608.1), SV40 promoter (DM063619.1), and then follow The above-designed sequence was added with the sequence GAATTC (EcoRI restriction site) at the 5' end and GAATTC (XhoI restriction site) at the 3' end, and then artificially synthesized; EcoRI and XhoI restriction digestion were inserted into the pUC19 plasmid, and the test was carried out. The plasmids successfully inserted into the above DNA fragments were then transfected into CHO cells (DG44). The sequences of the CMV promoter, CMV promoter TATA box, DHFR, BGHpA, SynpA, bGpA, and TKpA in...

Embodiment 3

[0049] Example 3 Expression and activity of anti-human platelet glycoprotein VI single-chain antibody (anti-GPVI scFv).

[0050] 3.1 Construction and expression of single-chain antibody expression vector

[0051] From blood (after transfusion of normal human blood) of patients with thrombocytopenic purpura (caused by deficiency of platelet collagen receptor GPVI) as a source, nucleated leukocytes (B lymphocytes) with affinity for platelets, namely The patient's B lymphocytes expressing GPVI antibodies were isolated. Then, using single-cell RT-PCR, specific primers for the variable region of the heavy chain and the variable region of the light chain of human immunoglobulin IgG, the DNA of the variable region of the light chain and the variable region of the heavy chain of the anti-GPVI antibody was cloned Sequence, the two are connected by DNA sequence; the Herceptin signal peptide DNA sequence is added before the light chain variable region sequence; GGATCC (BamHI restriction...

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Abstract

The present invention provides an expression vector for expressing proteins / polypeptides in mammalian host cells, comprising a marker gene and a restriction endonuclease cleavage site for inserting a target gene, and a Poly(A) tail after the target gene The number of signals is two or more, and the promoter element of the marker gene is only the TATA box. The upstream of the selectable marker gene of the expression vector provided by the present invention contains two or more Poly(A) tailing signals to minimize the interference of the strong upstream promoter on the activity of the downstream TATA box to drive the expression of the selectable marker gene. The expression vector of the present invention can generate stable and high-yielding cell lines for recombinant protein expression in a short time, and gene amplification does not require drug selection / mediation, and can be directly selected without any drug-induced amplification, These cell lines can stably express high levels of recombinant proteins for a long time and are very suitable for industrial production of proteins or polypeptides.

Description

technical field [0001] The invention belongs to the field of molecular biology and biomedicine, and specifically relates to an expression vector for expressing protein / polypeptide in mammalian cells, in particular to an expression vector for expressing recombinant immunotoxin which can be used for industrial production. Background technique [0002] Anticancer drugs is one of the largest and fastest growing markets in the pharmaceutical industry; the market is expanding globally and the demand for more effective and better-tolerated cancer therapies is increasing. The targeted drug market has been growing at a significant rate and there is great potential for continued growth in both developed and emerging regional markets. Monoclonal antibodies (Monoclonal antibodies, mAbs) have great advantages over small molecule (chemo) drugs (chemotherapy) in that they can recognize cancer cell surface markers with high specificity. As a result, mAbs often have fewer or milder side eff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/65
CPCC12N15/65C12N15/85
Inventor 孙冰
Owner 济南海湾生物工程有限公司
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