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A co-transfection eukaryotic expression vector for dhfr complementary expression and its preparation method and application

A eukaryotic expression vector and co-transfection technology, which is applied in the direction of using vectors to introduce foreign genetic material, cells modified by introducing foreign genetic material, fermentation, etc., can solve the problems of large differences in protein expression and different gene integration sites, etc. Achieve good application value

Active Publication Date: 2011-12-14
QILU PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The main problem of integrating foreign protein genes into cell chromosomes is that the gene integration sites are different, and the protein expression varies greatly

Method used

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  • A co-transfection eukaryotic expression vector for dhfr complementary expression and its preparation method and application
  • A co-transfection eukaryotic expression vector for dhfr complementary expression and its preparation method and application
  • A co-transfection eukaryotic expression vector for dhfr complementary expression and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construction of pCIneo-MWLD1 vector

[0042] The preparation method of the co-transfection eukaryotic expression vector of dhfr complementary expression, the steps are as follows:

[0043] (1) Digest the pCI-neo plasmid with restriction endonuclease SacII and BglII, insert the human β-globin nuclear matrix attachment region that has been double-digested with restriction endonuclease SacII and BglII with T4 ligase, and construct plasmid pCIneo -M;

[0044] (2) Plasmid pCIneo-M obtained in step (1) with restriction endonuclease SalI and NotI double digestion, insert the woodchuck hepatitis virus through restriction endonuclease SalI and NotI double digestion with T4 ligase Post-transcriptional regulatory sequence (WPRE), construct plasmid pCIneo-MW;

[0045] (3) Plasmid pCIneo-MW prepared in step (2) with restriction endonuclease MluI and XbaI double digestion, insert the ribosome entry site through restriction endonuclease MluI and XbaI double digestion with T4 ligase ...

Embodiment 2

[0058] Construction of complementary vectors expressing monoclonal antibodies

[0059] The gene fragment (SEQ ID NO.12) of the monoclonal antibody heavy chain encoding anti-EGFR2 and the gene fragment (SEQ ID NO.13) of the monoclonal antibody light chain of EGFR2 were double-digested with restriction endonucleases NheI and MluI respectively, and inserted into the Into the co-transfected eukaryotic expression vectors pCIneo-MWLD1 and pCIneo-MWLD2 cut with restriction endonucleases NheI and MluI, two complementary recombinant plasmids pCIneo-MHWLD1 and pCIneo-MLWLD2 were constructed.

[0060] Construction of Immunoglobulin Fusion Protein Complementary Vector

[0061] The gene fragment (SEQ ID NO.14) encoding TNFR: Fc fusion protein was double-digested with restriction endonucleases NheI and MluI respectively, and inserted into the co-transfection eukaryotic expression In vectors pCIneo-MWLD1 and pCIneo-MWLD2, two complementary recombinant plasmids pCIneo-MTWLD1 and pCIneo-MTWLD...

Embodiment 3

[0063] (1) Co-transfection of complementary expression eukaryotic vectors pCIneo-MHWLD1 and pCIneo-MLWLD2 to screen engineered cell lines

[0064] 1. Cell culture and transfection

[0065] The day before transfection, CHO cells were digested with trypsin and counted, and the CHO cells were plated on a 6-well plate so that the density reached 90% confluence on the day of transfection. CHO cells were plated in serum-containing, antibiotic-free DMEM medium. For each well of CHO cells, use 250 μl of OPTI-MEM I medium to dilute 5 μg of the pCIneo-MHWLD1 and pCIneo-MLWLD2 plasmids constructed in Example 2; for each well of CHO cells, use 250 μl of OPTI-MEM I medium to dilute 10 μl of LIPOFECTAMINE 2000 reagent. The diluted LIPOFECTAMINE 2000 was mixed with the diluted pCIneo-MHWLD1 and pCIneo-MLWLD2 plasmids within 30 minutes, and the mixture was incubated at room temperature for 20 minutes. Add the complex to each well and shake the plate to mix gently. At 37°C, 5% CO 2 After ...

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Abstract

The invention relates to an efficient eukaryotic expression vector capable of effectively expressing an antibody and other target proteins and a preparation method and an application thereof, belonging to the technical field of biotechnology. The vector is formed by inserting a nuclear matrix adhering zone, a marmot hepatitis virus post-transcriptional control sequence, a ribosome entry site sequence, a dhfr1-105aa gene segment or a dhfr106-187aa gene segment on a pCI-neo plasmid. According to the invention, the defects that time and energy are wasted when the traditional mammalian cells are expressed and screened and target protein expression level is low are overcome, and a vector transfection CHO (Chinese Hamster Ovary) cell applying the vector can obtain a monoclonal cell strain capable of stably and efficiently expressing antibodies or fusion proteins in a short time, thus the vector provided by the invention has good application value for efficient expression and industrialization of recombinant protein.

Description

technical field [0001] The invention relates to a eukaryotic expression vector, in particular to a high-efficiency eukaryotic expression vector capable of highly expressing antibodies and other target proteins, a preparation method and application thereof, and belongs to the technical field of biotechnology. Background technique [0002] At present, the development of eukaryotic cell expression systems represented by mammals is the development trend of the biopharmaceutical industry. Compared with the E. coli (prokaryotic cell) expression system, the expression of mammalian cells can not only ensure the correct pairing of disulfide bonds and protein folding of recombinant proteins, but also ensure the glycosylation of proteins, so that recombinant proteins expressed by mammalian cells It maintains a high degree of consistency with natural proteins in structure and function. In the drug market of developed countries, biological drugs expressed through mammalian cell culture ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N5/10C12P21/00
Inventor 王晶翼史文龙王庆民孙丽霞阎岩张莹宽陆贵全
Owner QILU PHARMA
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