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Gene expression system for producing recombinant protein by using CHO cells and eukaryotic expression vector

A technology of eukaryotic expression vectors and expression systems, applied in the field of gene expression systems and eukaryotic expression vectors, can solve the problems of technology transfer barriers restricting biopharmaceutical technology and industrial development

Inactive Publication Date: 2016-07-27
SHANGHAI HENLIUS BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Gene expression system for producing recombinant protein by using CHO cells and eukaryotic expression vector
  • Gene expression system for producing recombinant protein by using CHO cells and eukaryotic expression vector
  • Gene expression system for producing recombinant protein by using CHO cells and eukaryotic expression vector

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Experimental program
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Effect test

Embodiment 1

[0058] Construction of double gene expression vector pHLX101

[0059] Use primers S322 and S323 to linearize the expression vector of pHLX101-HLX01-HC (constructed by Shanghai Henlius Biotechnology Co., Ltd., see Chinese patent 201210211812.1), and use primers S324 and S325 to amplify The transcription unit of pHLX101-HLX01-LC (constructed by Shanghai Henlius Biotechnology Co., Ltd., see Chinese Patent 201210211812.1) expression vector, including promoter A sequence, HLX01 light chain gene sequence and SV40polyA gene sequence, and the introduction of enzyme cutting sites Spot BglII and XhoI.

[0060] Table 1. PCR primer sequence list

[0061]

[0062] Linearization pHLX101-HLX01-HCPCR reaction conditions: denaturation at 98°C for 10 seconds, annealing at 58°C for 5 seconds, extension at 72°C for 120 seconds, 35 cycles.

[0063] Reaction conditions for amplifying the transcription unit of the pHLX101-HLX01-LC expression vector: denaturation at 98°C for 10 seconds, annealin...

Embodiment 2

[0067] Optimized GS expression unit sequence

[0068] 1. Construction of pHLX101 double gene expression vector containing TK promoter and CHO cell GS gene

[0069] 1.1 GS gene sequence synthesis The front and back segments of the GS gene were prepared by chemical synthesis, wherein the front segment sequence was 1-1691 of SEQ ID No.3, and the latter segment was 1686-4599 of SEQ ID No.3. Using primers S341 (SEQIDNo.12) and S350 (SEQIDNo.13) and S342 (SEQIDNo.14) and S349 (SEQIDNo.15), PCR was used to amplify the front and back sections of the GS gene respectively. PCR reaction conditions: denaturation 98°C 5 seconds, annealing at 58°C for 5 seconds, extension at 72°C for 60 seconds, 35 cycles.

[0070] 1.2 Primers S355 (SEQ ID No. 16) and S337 (SEQ ID No. 18) for synthesizing the TK promoter sequence use the vector pHLX101 as a template to amplify the TK promoter. PCR reaction conditions: denaturation at 98°C for 5 seconds, annealing at 58°C for 5 seconds, extension at 72°C f...

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Abstract

The invention relates to the field of biotechnology, and in particular, relates to a gene expression system for producing a recombinant protein by using CHO cells and a eukaryotic expression vector. The invention provides the pHLX201 eukaryotic expression vector including a GS expression unit, the GS expression unit has the sequence comprising an SV40L promoter gene sequence and a glutamine synthetase gene sequence which are arrayed successively in a direction from 5' to 3', wherein the SV40L promoter gene sequence is shown in SEQ ID NO:2, and the glutamine synthetase gene sequence is shown in SEQ ID NO:3. The expression system provided by the invention can realize integration and efficient expression of exogenous genes in a CHO cell genome. The preparation method of the system mainly includes that the eukaryotic expression vector is constructed, is suitable for transient expression of a target gene in the CHO cells and is suitable for screening target gene high-expression cell lines. The CHO expression system can be used for realizing integration and expression of the recombinant protein, a monoclonal antibody and the like in the CHO cells, and has wide application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene expression system and eukaryotic expression vector for producing recombinant protein 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 expression has become th...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/52C12N5/10
Inventor 肖辉郎国竣姜伟东何虹霖
Owner SHANGHAI HENLIUS BIOTECH INC
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