Expression vector for animal cells, its preparation method and application

An expression vector and animal cell technology, applied in the field of genetic engineering, can solve the problem of low expression of the target protein, achieve the effect of increasing production and reducing translation efficiency

Active Publication Date: 2018-03-09
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to overcome the above disadvantages, provide an expression vector for animal cells, and solve the technical problem of low expression of the target protein in the host cell CHO in the prior art

Method used

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  • Expression vector for animal cells, its preparation method and application
  • Expression vector for animal cells, its preparation method and application
  • Expression vector for animal cells, its preparation method and application

Examples

Experimental program
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Embodiment 1

[0047] Embodiment 1 of the present invention provides an expression vector for animal cells, such as figure 1 As shown, the expression vector inserts the dhfr gene and the SV40-neo fragment into the pIRES vector digested by PvuII and BamHI, respectively, wherein the SV40-neo fragment is formed by connecting the SV40 early promoter gene and the neo gene, and the dhfr gene is located at Between the adjacent Xma I and Not I restriction sites; the SV40-neo fragment is located between the PvuII and BamHI restriction sites.

[0048] The above expression vector can be obtained by the following steps:

[0049] Step S101: using the pSV2-dhfr plasmid as a template and using SEQ ID No: 2 and SEQ ID No: 3 in the sequence table as primers for PCR amplification, wherein the primers shown in SEQ ID No: 2 contain an Xma I restriction site, The primer shown in SEQ ID No: 3 contains a Not I restriction site, and the pSV2-dhfr plasmid is from ATCC37146;

[0050] Step S102: The PCR product obta...

Embodiment 2

[0054] Embodiment 2 of the present invention provides an application of the expression vector of Embodiment 1, comprising the following steps:

[0055] Step S201: Insert the target gene into the multiple cloning site of the expression vector obtained in Example 1 to obtain a recombinant vector;

[0056] Step S202: using dhfr-deficient CHO cells as hosts to transfect expression vectors;

[0057] The day before transfection, inoculate 1X106 dhfr-deficient CHO cells in a 60mm culture dish, add 5ml serum-containing, antibiotic-free DMEM medium, 37°C, 5% CO 2 Culture until the cells are required to be 40%-80% confluent at the time of transfection. Dilute 5 μg of recombinant plasmid with OPTI-MEM1 medium to a total volume of 250 μl. After dilution, invert the centrifuge tube several times to mix the mixture. Add 10 μl transfection reagent LIPOFECTAMINE2000 to the mixture, and pipette several times with the pipette. Incubate the mixture at room temperature for 5-10 min. While inc...

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Abstract

The present invention relates to the field, in particular to an expression vector for animal cells, which contains a strong expression inducible promoter, a multiple cloning site for gene integration, a ribosome entry site sequence, a dihydrofolate reductase gene, and SV40 early promoter gene, neo gene and drug resistance gene with enhancer. The present invention also provides the preparation method, recombinant vector and application of the above expression vector. In the expression vector of the present invention, a promoter without an enhancer is used to weaken the expression of the neo gene, and the dhfr gene is located downstream of the ribosome entry site IRES as a co-amplified gene, thereby reducing the translation efficiency of the dhfr gene, combined with G418 pressure selection and Dihydrofolate reductase gene stress screening to increase the expression of the target protein.

Description

【Technical field】 [0001] The invention relates to the technical field of genetic engineering, in particular to an expression vector for animal cells, a preparation method thereof and the application of the expression vector to high-level expression of a target protein in eukaryotic cells. 【Background technique】 [0002] In the process of protein synthesis, it must undergo post-translational processing and modification to become an active protein. Among them, the post-translational processing process includes: phosphorylation, glycosylation, disulfide bond formation, acylation and protease processing. Prokaryotic cells do not have the ability of post-translational processing, therefore, prokaryotic cells are not effective in expressing eukaryotic proteins. Although yeast, plant, and insect cells can glycosylate recombinant proteins, their glycosylation methods are different from those of mammalian cells, which may cause immunogenicity. Therefore, it is an urgent task in this...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/66
Inventor 万晓春李俊鑫赵琦王蒲
Owner SHENZHEN INST OF ADVANCED TECH
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