Method for constructing stable cell strain secreting expression protein

A technology of secretion expression and construction method, which is applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., which can solve the problems of heavy workload and difficult purification, and achieve low cost and low reagent cost , the effect of less impurity protein
CN107043786APending Publication Date: 2017-08-15合肥知恩生物技术有限公司
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
合肥知恩生物技术有限公司
Publication Date
2017-08-15

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Abstract

The invention belongs to the field of biotechnology and particularly relates to a method for constructing a stable cell strain secreting an expression protein. The method comprises the following steps: with a recombinant lentiviral vector pCDH-S-His as a skeleton, constructing a target protein gene to the recombinant lentiviral vector pCDH-S-His to obtain a recombinant plasmid pCDH-S-P-His; co-transfecting the recombinant plasmid and helper plasmids pSPAX2 and pMD2.G with 293T cells by using a PEI transfection method to obtain a recombinant lentivirus; transducing the obtained lentivirus to the 293T cells, and performing puromycin screening to obtain a plurality of monoclonal cell strains; detecting the content of target protein in each cell supernatant by an Elisa method; determining a high-expression cell strain. Compared with a traditional plasmid transfection method, the method provided by the invention has the advantages of short experimental period, high positive rate and good stability of the cell strain.
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Description

technical field

[0001] The invention belongs to the field of biotechnology, in particular to a method for constructing a stable cell line that secretes and expresses proteins. Background technique

[0002] The construction of eukaryotic expression plasmids, which can be transfected into 293T cells, can realize the transient expression of proteins, which is currently the most commonly used eukaryotic protein expression method. However, if a large amount of target protein needs to be expressed, a large amount of plasmid extraction, cell culture, transfection, broken cells are required to extract the total protein, and then purified, which is a heavy workload and difficult to purify.

[0003] Simultaneous co-transfection of lentiviral expression vector and packaging plasmid into 293T cells can produce high-titer virus particles. Then, the virus acts on the host cell, so that the target gene can enter the host cell, undergo reverse transcription, and integrate into the genome, ...

Claims

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