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Eukaryotic expression vector for producing recombinant protein by using CHO cells, and system

A technology of eukaryotic expression vector and expression system, applied in the field of gene expression system and eukaryotic expression vector, can solve the problems of technology transfer barriers restricting biopharmaceutical technology and industrial development, etc.

Active Publication Date: 2014-01-15
SHANGHAI HENLIUS BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The expression vectors using GS and DHFR gene amplification and screening systems are mostly developed by developed countries such as the United States. Their patent protection and technology transfer barriers have greatly restricted the development of my country's biopharmaceutical technology and industry. expression system to promote the development of related protein drugs and the progress of my country's biopharmaceutical industry

Method used

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  • Eukaryotic expression vector for producing recombinant protein by using CHO cells, and system
  • Eukaryotic expression vector for producing recombinant protein by using CHO cells, and system
  • Eukaryotic expression vector for producing recombinant protein by using CHO cells, and system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Construction of pHLX101 eukaryotic expression vector

[0056] 1. Synthesis of Marker Gene Expression Units

[0057] The mouse glutamine synthetase gene sequence (GS, Genebank accession number: NM 008131.3, ABC015086.1, X16314.1), TK promoter gene sequence (TK promoter, Genebank accession number: JN420340. 1, AF104248.2, AF362551.1) and SV40polyA gene sequence (Genebank accession number: JQ302818.1, HQ388295.1, EF437954.1), based on these sequences, designed a GS that facilitates the integration and expression of the GS gene in CHO cells Expression unit (SEQ ID NO: 4), the designed expression unit was sent to Gene Synthesis Company to synthesize the sequence, and the synthesized expression unit was inserted into the pBSK vector to obtain the TK-GS-SV-pBSK vector.

[0058] 13. Construction of eukaryotic expression vector pHLX101

[0059] 2.1 Preparation of gene fragments containing GS expression units

[0060] The gene fragment containing the GS expression un...

Embodiment 2

[0082] Example 2 Construction of CHO cell gene integration expression system and expression of antibody

[0083] 1. Construction of pHLX101-HLX01-HC, pHLX101-HLX01-LC recombinant plasmids and CHO expression system

[0084] Firstly, the eukaryotic expression vector 2009-HLX01-HC (containing the HLX01-HC expression unit composed of PromoterA, antibody heavy chain sequence and SV40 poly A) containing antibody heavy chain and antibody light chain sequences and related functional elements was constructed, and the true Nuclear expression vector 2009-HLX01-LC (contains HLX01-LC expression unit composed of CMVPromoter, antibody light chain sequence and SV40polyA).

[0085] The exogenous gene expression units were respectively amplified from the 2009-HLX01-HC vector and the 2009-HLX01-LC vector by PCR, and then the exogenous gene expression units were inserted into the pHLX101 vector by enzyme digestion and ligation, and transfected CHO-K1 cells, and finally screen the cell line with ...

Embodiment 3

[0143] Example 3 Construction of CHO cell gene integration expression system and expression of foreign protein

[0144] 1. Construction of pHLX101-eGFP expression vector and screening of stable cell lines

[0145] 1.3. Expression vector construction

[0146] First, construct the eGFP expression unit, which contains the SmaI endonuclease recognition site sequence and CMV promoter sequence in the 5' to 3' direction (the gene sequence is the same as the Genebank login sequence: JQ302818.1, AB609714.1, GU937742.1, BK000394.5 and other sequences), eGFP gene sequence (the gene sequence is the same as the Genebank landing sequence: JQ809330.1, JN038403.1, JQ733047.1, AB673329.1 and other sequences), BGHpA sequence (the gene sequence is the same as the Genebank landing sequence: EF437956.1 , U90717.1, X90639.1, JF313342.1 and other sequences) and SmaI endonuclease recognition site sequence. The exogenous gene expression unit was prepared by digesting the eGFP expression unit with Sm...

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Abstract

The invention relates to the field of genetic engineering, and specifically discloses a gene expression system for producing recombinant protein by using CHO cells, and an eukaryotic expression vector, wherein the eukaryotic expression vector comprises a GS expression unit, and the GS expression unit comprises a TK promoter gene sequence aligned along a 5' to 3' direction, a glutamine synthetase gene sequence and a SV40polyA gene sequence. The expression system comprises the eukaryotic expression vector and CHO cells. With the expression system, integration and efficient expression of exogenous gene in CHO cell genome can be achieved, and broad application prospects are provided in the field of protein.

Description

technical field [0001] The invention relates to the field of genetic engineering, and specifically discloses a gene expression system and a eukaryotic expression carrier for producing recombinant proteins using CHO cells. Background technique [0002] Gene expression systems used in the field of genetic engineering are broadly classified into prokaryotic, yeast, plant, insect and mammalian cell expression systems. Compared with other systems, the advantage of mammalian cell expression system is that it can guide the correct folding of proteins and provide complex glycosylation modifications, so the expression products are closest to natural higher organisms in terms of molecular structure, physical and chemical properties and biological functions. protein molecule. The large-scale production technology of mammalian cells is also becoming more and more mature and perfect. Therefore, the preparation of therapeutic recombinant protein drugs through mammalian cell culture expr...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12P21/00C12R1/91
Inventor 姜伟东郎国竣王彦玲陈莹刘世高郭新军马辰张二辉
Owner SHANGHAI HENLIUS BIOTECH INC
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