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Construction and application of recombinant human interleukin 12 eukaryotic expression vector

A technology of eukaryotic expression vector and interleukin, which is applied in application, recombinant DNA technology, medical preparations containing active ingredients, etc., can solve the problems that it cannot be used in clinical use and the purity of rhIL-12 is not enough

Inactive Publication Date: 2010-02-03
张文卿
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Problems solved by technology

However, this method produces rhIL-12, and the purity of the produced rhIL-12 is not enough, so it cannot be used in clinical use at present.

Method used

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Embodiment Construction

[0017] This specific embodiment adopts the following technical scheme: construction of pCDNA6-IL-12: the fusion gene of rIL-12P40 and P35 is obtained by connecting 10 hydrophobic flexible amino acid linkers. The ratio of 1:1 is expressed, and the flexible amino acid linker can make the two subunits fully extended and folded correctly, so as to obtain the maximum biological activity. The fusion gene was inserted into the eukaryotic expression plasmid (pcDNA6 / v5-hisp) to construct pCDNA6-IL-12; the recombinant clone was identified by enzyme digestion and sequencing. pCDNA6-IL-12 was introduced into HEK293 cells, and the expression of rhIL-12 in the culture supernatant was detected by ELISA.

[0018] In vitro biological activity analysis of rh-IL-12: The in vitro biological activity of rhIL-12 was analyzed by specific lymphocyte proliferation assay, NK killing activity, cytokine flow cytometry (CFC) and other methods.

[0019] Gene adjuvant function study of pCDNA6-IL-12: pCDNA6...

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Abstract

The invention provides a construction and an application of recombinant human interleukin 12 eukaryotic expression vector and relates to the development of recombinant human interleukin 12 (rhIL-12) eukaryotic expression vector, the screening of stable expression cell lines and the adjuvant function of nucleic acid vaccine gene and the therapeutical effect of tumor gene. Ten hydrophobic flexible amino acid joints are used to connect P40 and P35 to obtain fusion gene to construct rhIL-12 eukaryotic expression plasmid (pCDNA6-IL-12), then the expression plasmid is transferred to CHO cell and finally stable expression cell lines are screened. The expression product has good biological activity. Nucleic acid vaccine and pCDNA6-IL-12 are used for coimmune so as to significantly increase the immune effect. Strong antitumor effect can be exerted when the expression vector of the invention is directly injected inside the tumor of laboratory mouse tumor model or is first used to perform gene modification to tumor cells and then implanted in tumor. The expression vector of the invention can be used in the search of clinical trial stage after further perfecting.

Description

Technical field: [0001] The invention relates to the development and application of recombinant human interleukin 12 (rhIL-12) eukaryotic expression vector. It specifically relates to the construction of the carrier, expression identification, biological activity analysis, screening of rhIL-12 stably expressing cell lines in eukaryotic cells, a gene adjuvant for nucleic acid vaccines and tumor gene therapy preparations. Background technique: [0002] IL-12 is a cytokine composed of heterodimeric proteins. The genes encoding the two subunits (p35 and p40) of IL-12 are located on different chromosomes. Sulfur bonds into a biologically active dimeric molecule (p70). Individual subunits cannot exert their biological role. Moreover, p40 tends to form homodimers and inhibit the activity of IL-12 through a receptor competition mechanism. [0003] Chinese patent 200410080518.7 discloses a method for purifying recombinant human interleukin-12, which relates to a protein purificati...

Claims

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Application Information

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IPC IPC(8): C12N15/24C12N15/85C12N5/10A61K39/39A61K48/00A61P35/00
Inventor 张文卿
Owner 张文卿
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