Efficient screening method of exogenous protein expression cell strain

A screening method and exogenous protein technology, which is applied in the field of exogenous protein expression cell line screening, can solve the problems of time-consuming and labor-consuming, complicated cell line screening process, etc.

Inactive Publication Date: 2015-03-11
SHANGHAI MBR BIOMEDICAL TECH
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Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of complicated, time-consuming and labor-consuming screening

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  • Efficient screening method of exogenous protein expression cell strain
  • Efficient screening method of exogenous protein expression cell strain
  • Efficient screening method of exogenous protein expression cell strain

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Embodiment Construction

[0029] Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art, which will be understood by those skilled in the art.

[0030] Referring to, for example, standard manuals, the practitioner is referred to standard texts and commentaries on cell biology, histology and embryology for further details on conventional techniques employed in the practice of the present invention. Including Teratocarcinomas and embryonic stem cell: A practical approach [edited by E.J.Robertson, IRL Publishing Co., 1987]; Guide to techniques in Mouse Development [edited by P.M.Wasserman et al., Academic Press, 1993]; Embryonic Stem Cell Differentiation in Vitro [M.V. Wiles, Meth. Enzymol. 225: 900, 1993]; Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy [P.D. Rathjen et al., Reprod. Fertil. Dev. 10: 31, 1998].

[0031] Cell biology, protein chemistry, and antibody technology can be found in "Current Protocols ...

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Abstract

The invention belongs to the technical field of cell engineering, and particularly relates to an efficient screening method of an exogenous protein expression cell strain. The efficient screening method of the exogenous protein expression cell strain disclosed by the invention comprises the following steps: using an interest protein expression vector to transfect host cells; pressurizing a screening drug, and forming a stable cell pool; inoculating a semisolid culture medium with the cells in the stable cell pool; transferring the cloned cells from the semisolid culture medium to a 96-hole cell plate to be continuously cultured for 4-5 days, and detecting the interest protein expression index in the cell supernatant; transferring cells which are first 50 in the ranking of expression index to a 24-hole plate to be continuously cultured for 5 days; further transferring the cells in the 24-hole plate to a 6-hole to be continuously cultured for 5 days; transferring the cells in the 6-hole plate to a shake flask for culture, and detecting and evaluating the interest protein expression indexes of different cloned cells under a suspension state; according to the evaluation result in the shake flask, performing a stability passage test on the cells which are first 10 in the ranking of the expression index; according to the interest protein expression level of the cell strain and the stability of the continued expression protein, determining the candidate cell strains.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a high-efficiency screening method for exogenous protein expression cell lines. Background technique [0002] In the entire biopharmaceutical industry, about 70% of protein drugs are expressed by Chinese hamster ovary cells (CHO). [0003] The construction technology of cell lines expressing foreign proteins has always been one of the core technologies of major biopharmaceutical companies. Traditional cell screening methods have many steps (see figure 1 ), specifically: 1. The target protein expression vector is transfected into CHO cells. 2. Screening drugs to pressurize and stabilize the formation of cell pools. 3. The stable cell pool cells were inoculated into 96-well cell culture plates by limiting dilution method to form a monoclonal cell population. 4. Expand the cells in the 96-well plate to the 24-well plate. 5. The expression level of the 24-we...

Claims

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Application Information

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IPC IPC(8): C12N15/85
Inventor 陈亮王佳龙都业杰
Owner SHANGHAI MBR BIOMEDICAL TECH
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