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Mammal cell expression system

A mammalian cell technology, applied in the field of efficient and stable mammalian cell expression system, can solve the problems of inconvenient screening work and achieve the effects of reducing the probability of false positive clones, reducing the size of the vector, and reducing the burden

Active Publication Date: 2014-07-23
UNICOHEALTH CO LTD
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  • Abstract
  • Description
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Problems solved by technology

In addition, due to the non-specific integration of exogenous genes, the large size of the vector will cause its elements to integrate into different positions of the host cell genome, which will bring great inconvenience to the later screening work

Method used

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  • Mammal cell expression system
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Construction of mammalian cell expression vector

[0044] Methods: The traditional expression vector pA was constructed by molecular biology techniques, such as restriction enzyme digestion, DNA ligation, Escherichia coli transformation and clone screening, according to the standard methods of "Molecular Cloning Experiment Guide" (third edition, translated by Huang Peitang, Science Press) And the high expression vector pB of the present invention. The specific operation method is as follows: Using the pcDNA3.1 vector of Invitrogen Company as a template, artificially synthesize the fusion sequence (1.8 kb) of EF1-α promoter, DHFR, HSV TK terminator and other regulatory sequences, and add SpeI and NaeI at the end respectively. Restriction sites. The synthetic fusion sequence was connected to pcDNA3.1 double-digested by SpeI and NaeI using T4DNA ligase, transformed into Escherichia coli, and carried out single clone screening to obtain the traditional vector...

Embodiment 2

[0069] Embodiment 2: the comparison of the expression amount of EPO protein in the traditional expression system and the expression system of the present invention

[0070] 2.1. Construction of an expression vector containing the target gene: artificially synthesize the coding sequence of EPO according to the existing sequence information of NCBI, and clone it into the expression vector constructed in Example 1 by introducing corresponding restriction sites (NheI and HindIII) Among pA and pB, the expression vectors pA-EPO and pB-EPO containing the target gene were obtained.

[0071] 2.2. DNA transfection into CHO-dhfr - Suspension cell lines: Transfect the above-mentioned expression vectors pA-EPO and pB-EPO containing the target gene, and expression vector control pA and pB into DHFR enzyme-deficient CHO cells (CHO-dhfr - )middle. CHO-dhfr - The cells were cultured in suspension, and the cell density was 1.5×10 6 / m], the transfection volume was 30ml. The transfection me...

Embodiment 3

[0084] Embodiment 3: the comparison of the expression amount of EPO protein in the expression system containing different promoter vectors

[0085] 3.1. Experimental method: the experimental method of constructing the expression vector containing the target gene, DNA transfection into CHO-dhfr - The method for screening suspension cells and stable cells is the same as that in Example 2. In order to construct expression vectors containing different promoters before the target gene, the CMV promoters in the expression vector pB containing the EPO target gene in Example 2 were replaced with artificially synthesized MPSV promoters and RSV promoters respectively, and then the above-mentioned constructed The expression vector pB-EPO containing different promoters and EPO were transfected into DHFR enzyme-deficient CHO cells (CHO-dhfr - ), a stable expression cell line was obtained after initial screening with Zeocin selection medium and MTX gradient pressurization screening.

[00...

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Abstract

The invention relates to a high-efficiency stable mammal cell expression system. The high-efficiency stable mammal cell expression system efficiently expresses a target gene in an appropriate mammal host cell strain by applying a novel high-efficiency expression vector. The high-efficiency stable mammal cell expression system disclosed by the invention can outstandingly enhance the expression level of the target gene in a host cell, greatly reduce the screening time and cost of a high-expression cell strain and realize the high-yield stable expression of a target protein in a mammal cell.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an efficient and stable mammalian cell expression system, which can realize high-efficiency and stable expression of an exogenous target protein in mammalian cells by using the expression system of the invention. Background technique [0002] Therapeutic recombinant proteins (including antibodies) have been widely used clinically. Many therapeutic recombinant protein drugs, such as human tissue plasminogen activator, erythropoietin (Erythropoietin, EPO for short), coagulation factor VIII and monoclonal Antibody CD20 often requires specific post-translational modifications (such as glycosylation) in order to maintain its biological activity, while prokaryotic or low-level eukaryotic cell expression systems lack complex post-translational modification functions, so mammalian expression systems, especially CHO Cell (Chinese hamster ovary, Chinese hamster ovary cells) express...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85
Inventor 侯永敏李强廖莎雷瑶
Owner UNICOHEALTH CO LTD
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