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Construction and application of mammal cell high-efficiency expression vector

A eukaryotic expression vector and vector technology, which can be applied to cells modified by the introduction of foreign genetic material, the use of vectors to introduce foreign genetic material, recombinant DNA technology, etc., which can solve the problem of multiple clones and the difficulty of screening high-expression cell lines, and the cost is very long. Time and other issues to achieve the effect of improving efficiency, reducing low-expression background clones, and overcoming position effects

Active Publication Date: 2015-01-21
BIOTECH PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that: in CHO cells, the transcriptional activity of the CMV promoter is about 10 times and 30 times that of the SV40 promoter and the LTR (Rous sarcoma virus gene) promoter, respectively. In previous studies, we passed a relatively strong promoter A promoter such as CMV promoter to drive the expression of the target gene, and a weaker promoter such as SV40 promoter to express the screening gene, but there are still many clones formed in the end, and high-expression cell lines are selected from thousands of transfectants is still difficult and often takes a long time

Method used

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  • Construction and application of mammal cell high-efficiency expression vector
  • Construction and application of mammal cell high-efficiency expression vector
  • Construction and application of mammal cell high-efficiency expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of the pTSE-w vector and verification of the validity of the WPRE sequence

[0044] 1, Construction of pTSE-w vector:

[0045] The WPRE sequence is shown as SEQ IN NO: 3, and was commissioned to be synthesized by Shanghai Jierui Bioengineering Co., Ltd. Design PCR primers for amplification, and then digest with BamHI and BglII to obtain the target fragment.

[0046] P1: 5'-TAG GGATCC AATCAACCTCTGGA-3'

[0047] P2: 5'-TGT AGATCT CGAAGACGCGGAAGAGGCCG-3'

[0048] Linearize the pTSE vector with BamHI and dephosphorylate it to prevent the self-ligation of the vector; because BamHI and BglII are homologous enzymes, the sticky ends generated can be ligated, and finally pick the pTSE-w vector whose WPRE insertion direction is forward (we It is specified that the same insertion direction as PCMV-MCS-RBGpolyA is forward). At this time, the BamHI site of the original multiple cloning site is retained, and after the WPRE sequence is connected, th...

Embodiment 2

[0059] Example 2: Construction of pSNEO vector and verification of high efficiency in cell line construction

[0060] 1. Construction of pSNEO vector

[0061] Based on the pTSE-w vector, construct the vector pSNEO for screening stably transfected cell lines. The specific construction method is as follows: Use PcDNA3.1 + The plasmid (purchased from Invitrogen) was used as a template, and the synthetic primer sequences were as follows (PNEOF-1 and PNEOR-2 synthesized 5' phosphorylated primers):

[0062] PNEOF-1: 5'-GCAGGCAGAAGTATGCAAAG-3'

[0063] PNEOR-1: 5'-GATGTCTACTGCATCCTCGATGTGATCAGATCCGAAAAT-3'

[0064] PNEOF-2: 5'-AGGATGCAGTAGACATCCTCGATGATTGAACAAGATGGAT-3'

[0065] PNEOR-2: 5'- GATATC GCT ACATGT ATACAGACATGATAAGATACATTGATG-3'

[0066] The part marked in the box is the reverse complementary sequence, the underlined part is the restriction site of EcoRV and PciI respectively, the fragment NEO1 is obtained by the amplification of PNEOF-1 and PNEOR-1, the fragme...

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Abstract

The present invention relates to a mammalian cell high-efficiency expression vector pSNEO, which is constructed based on the neomycin resistance screening gene NEO by combining the strategy of weakening the expression of the screening gene and enhancing the expression of the target gene. Wherein, the attenuation of the screening gene includes two aspects, the introduction of a deletion mutation in the promoter to achieve the attenuation of the transcription of the screening gene, and then the introduction of a hairpin structure sequence before the translation start site of the screening gene to achieve attenuation of the translation of the screening gene. To enhance the expression of the target gene, a transcriptional regulatory element WPRE sequence is added between the multiple cloning site of the target gene expression frame and the PolyA tailing signal. The pSNEO vector constructed by the invention can conveniently and quickly complete the construction of a stable high-expression cell line of the target gene, and provides a new tool for the preparation of macromolecular recombinant proteins.

Description

technical field [0001] The invention relates to biopharmaceutical gene engineering expression technology, in particular to the construction and application of a mammalian cell high-efficiency expression vector. Background technique [0002] The acquisition of high-efficiency mammalian cell expression system and high-expression engineered cell line is the bottleneck in the research and production of genetically engineered drugs, and expression vector and host cell are the two basic components of mammalian cell expression system. The expression systems currently used to prepare genetically engineered drugs include prokaryotic expression systems with Escherichia coli as hosts and eukaryotic expression systems with yeast, insect cells, plant cells and mammalian cells as hosts. [0003] The CHO expression system is one of the most widely used eukaryotic expression systems and one of the few animal cells that can be cultured in high-density suspension. The exogenous gene product ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/64C12N15/66C12N5/10
Inventor 孟艳敏刘志刚王康任秀梅高震郭晶晶彭博白先宏
Owner BIOTECH PHARMA CO LTD
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