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Expression vector of mammalian cell for industrial production

An expression vector and mammalian technology, applied in the fields of molecular biology and biomedicine, can solve the problems of difficulty in the optimization process, failure to screen high-expression cell lines, and high cost, and achieve the effect of avoiding read-through

Active Publication Date: 2018-07-24
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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  • Expression vector of mammalian cell for industrial production
  • Expression vector of mammalian cell for industrial production
  • Expression vector of mammalian cell for industrial production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Construction of vector

[0041] The sequences of the CMV promoter and its TATA box, mouse DHFR, BGH polyA (BGHpA), beta-globinpolyA (bGpA), TK polyA (TKpA) were obtained through GenBank, and then GAATTC (EcoRI restriction site) was sequenced from 5' to 3' dot)-CMV promoter-Kozak sequence-BamHI and SalI restriction sites-BGHpA-SynpA-bGpA-CMV TATA box-DHFR-TKpA-GAATTC (XhoI restriction site) after the sequence arrangement, artificially synthesized as SEQ The DNA fragment shown in No. 5; EcoRI and XhoI digested and inserted into the pUC19 plasmid, and the plasmid that was successfully inserted into the above DNA fragment after testing is the vector for protein expression in mammalian cells.

Embodiment 2

[0042] Example 2 Expression of GFP (Green Fluorescent Protein).

[0043] 2.1 Construction and expression of the vector

[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 sequence designed above is artificially synthesized by adding the sequence GAATTC (EcoRI restriction site) at the 5' end and GAATTC (XhoI restriction site) at the 3' end; after EcoRI and XhoI restriction restriction, insert it into the pUC19 plasmid and check The plasmids successfully inserted with the above DNA fragments were transfected into CHO cells (DG44). The sequences of the CMV promoter, CMV promoter TATA box, DHFR, BGHpA, SynpA, bGpA, and TKpA in the vector were as in Example 1.

[0...

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 scFv expression vector

[0051] From the blood of patients with thrombocytopenic purpura (caused by platelet collagen receptor GPVI deficiency) (after transfusion of normal human blood) as a source, nucleated white blood cells (B lymphocytes) with affinity for platelets are isolated, namely The patient's B lymphocytes expressing GPVI antibodies were isolated. Then, using single-cell RT-PCR and specific primers for the heavy chain variable region and light chain variable region of human immunoglobulin IgG, clone the DNA of the light chain variable region and heavy chain variable region of the anti-GPVI antibody sequence, the two are connected by DNA sequence; add Herceptin signal peptide DNA sequence before the light chain variable region sequence; Carry out artificial synthesis, then insert into the vector obtaine...

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Abstract

The invention provides an expression vector for expressing protein / polypeptide by a mammalian host cell expression protein / polypeptide. The expression vector contains a marker gene and a restriction-endonuclease enzyme digestion site, wherein the restriction-endonuclease enzyme digestion site is used for being inserted into insertion of a target gene; the quantity of Poly(A) tailing signals behindthe target gene is 2 or more than 2; a starting component of the marker gene is only a TATA box. The upstream end of the selectable marker gene of the expression vector provided by the invention contains two or more Poly(A) tailing signals; the interference, to the activity of the downstream TATA box, of an upstream strong promoter is minimized, so as to drive the expression of the selectable marker gene. The expression vector provided by the invention can be used for generating stable and further high-yield cell lines for the expression of recombinant protein within a short time; moreover, the gene amplification does not need drug selection / medication; the selection can be directly carried out in the condition that any no drug induced amplification does not exists, and these cell lines can be used for stably expressing high-level recombinant protein for a long time, and is quite applicable to the industrialized production of the protein or the polypeptide.

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 Applications(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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