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CHO cell line, construction method, recombination protein expression system and application

A construction method and technology for recombinant proteins, applied in the direction of recombinant DNA technology, biochemical equipment and methods, and other methods for inserting foreign genetic materials

Active Publication Date: 2020-07-07
XINXIANG MEDICAL UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the current research, there is no report that APRT-deficient cell lines were obtained by knocking out the APRT gene of CHO cells through CRISPR / Cas9 gene editing technology, and then used for efficient and stable expression of recombinant proteins

Method used

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  • CHO cell line, construction method, recombination protein expression system and application
  • CHO cell line, construction method, recombination protein expression system and application
  • CHO cell line, construction method, recombination protein expression system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of APRT gene-deficient CHO cell line

[0042] In this embodiment, the APRT gene-deficient CHO cell line, its construction method comprises the following steps:

[0043] 1. Determine the target site for the candidate gene

[0044] 1) Amplify the partial sequence of the ARPT gene:

[0045] Amplification primers were designed according to the APRT genome sequence (No.X03603.1, shown in SEQ ID NO.1) in GenBank of NCBI:

[0046] APRT-PCR-L: 5'-CCAGGCTTTCAATTTGAGGT-3' (as shown in SEQ ID NO.2);

[0047] APRT-PCR-R: 5'-ACTCATCCAGGGTCAACGAG-3' (as shown in SEQ ID NO.3);

[0048] Perform PCR amplification on the APRT gene fragment, clone and sequence the PCR amplified fragment to verify the accuracy of the sequence, and the verified amplified sequence is shown in SEQ ID NO.4.

[0049] 2) Determine the sgRNA targeting site sequence:

[0050] The sequence of the sgRNA targeting site of the APRT gene assisted by the online tool (http: / / crispr.mit.edu / ) i...

Embodiment 3

[0073] Example 3 Using the APRT gene-deficient CHO cells constructed in Example 1 of the present invention to carry out the expression system construction and expression analysis of the target gene eGFP

[0074] 1. Construction of APRT weakening vector pWTY3G-APRT-EGFP-mut

[0075] The present invention takes the eukaryotic expression vector pIRESneo2 (Clontech company) as the base carrier, and constructs the eukaryotic expression vector pWTY3G-EGFP driven by the EF-1α promoter to express green fluorescent protein (eGFP) (see image 3 ), whose sequence is shown in SEQ ID NO.7. Synthesize the APRT gene expression cassette sequence driven by the weakened SV40 promoter and the start codon mutation (as shown in SEQID NO.8) and insert it into the upstream of the basic vector EF-1α promoter. The constructed eukaryotic expression vector can be simultaneously The expression of eGFP and weakened APRT is named pWTY3G-APRT-EGFP-mut, and its sequence is shown in SEQ ID NO.9.

[0076] 2....

Embodiment 4

[0084] Example 4 Using the APRT gene-deficient CHO cells constructed in Example 1 of the present invention to carry out the expression system construction and expression analysis of the target gene vitronectin (vitronectin, VTN)

[0085] The weakened APRT expression vector constructed in Example 3 above is pWTY3G-APRT-EGFP-mut as the base vector, and the eGFP sequence in the base vector is replaced with the vitronectin (VTN) sequence (as shown in SEQ ID NO.10) to construct The vector pWTY3G-AP / Vitin-M expressing VTN and weakened APRT, the control vector is the vector pWTY3G-VTN expressing only VTN without the weakened APRT expression cassette.

[0086] The constructed expression vector plasmids were respectively transfected into APRT gene-deficient CHO cells and normal CHO cells constructed in Example 1 of the present invention. The transfected cells were cultured in a medium containing G418 (800 μg / mL) for two weeks to select stably transfected recombinant cell pools, and eac...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a CHO cell line, a construction method, a recombination protein expression system and an application. The APRT gene defective Type CHO cell line is used for constructing the recombination protein expression system, and no matter whether G418 screening pressure exists or not, the expression and maintaining rate ofa target protein eGFP in the recombination protein expression system constructed with 60th generation APRT gene defective type CHO cells is far higher than that of eGFP in normal recombination CHO cells. When the G418 screening pressure does not exist, the expression level of recombination vitronectin in the 30th generation cells of the recombination protein expression system constructed by the CHO cells is notably higher than that of corresponding target proteins in normal CHO cells. The recombination protein expression system can notably increase the expression level and long-term expression stability of the target gene in the CHO cells, and the problem that present CHO cell expression system is unstable in expression, can be solved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a CHO cell line, a construction method, a recombinant protein expression system, and an application. Background technique [0002] The Chinese hamster ovary (CHO) cell expression system is currently an important platform for the research and development and production of biomedicine such as medicinal recombinant proteins (antibodies), and is currently the most widely used mammalian cell expression system. CHO cells have the advantages of easy genetic modification and expansion, easy transfection, correct protein folding and glycosylation modification, secreted expression and easy purification of products, suspension culture, high cell density, and large-scale production. . In the industrial production of recombinant drug proteins, from the CHO recombinant expression seed cell bank to the scale of large-scale bioreactors, the cells need to be stably subcultured for at...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/90C12N9/22
CPCC12N9/1077C12Y204/02007C12N15/85C12N15/907C12N9/22C12N15/1137C12N2310/20C12N15/113
Inventor 贾岩龙王天云路江涛杨亮倪天军王小引郭潇林艳王冲
Owner XINXIANG MEDICAL UNIV
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