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Construction method and application for stably expressing HPV16 E5 protein cell strain

A technology for HPV16E5 and stable expression, which is applied in the field of construction of cell lines that stably express HPV16E5 protein, which can solve the problems of high hydrophobicity, weak antigenicity, and research obstacles.

Inactive Publication Date: 2016-03-23
BEIJING UNIV OF TECH
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  • Abstract
  • Description
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Problems solved by technology

Due to the high hydrophobicity and weak antigenicity of E5 protein, and there is currently no corresponding antibody, the research on this protein has been hindered

Method used

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  • Construction method and application for stably expressing HPV16 E5 protein cell strain
  • Construction method and application for stably expressing HPV16 E5 protein cell strain
  • Construction method and application for stably expressing HPV16 E5 protein cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0013] specific implementation plan

[0014] (1) Construction of recombinant vector

[0015] (1) HPV16E5 gene amplification

[0016] According to the whole genome sequence of HPV16 (GenBankNC_001526), ​​the HPV16E5 specific primers with His tag, EcoR1 and BamH1 double restriction sites and protective bases were designed. '-CGCGGATCCTTAATGATGATGATGATGATGTGTAATTAAAAAGCGTGCATGT-3' (SEQ ID NO. 2). The PCR reaction system is:

[0017]

[0018]

[0019] (2) DNA agarose gel electrophoresis

[0020] Prepare 1.5% agarose, and heat it in a microwave oven to completely dissolve the agarose particles; place the washed and dried gel plate horizontally on the workbench; mix well, fill the gel, and insert a comb to avoid air bubbles; wait for the gel to solidify , carefully pull out the comb; put the gel into the electrophoresis tank, add the electrophoresis buffer; load the PCR product, and electrophoresis at 110V for 30min.

[0021] (3) PCR product gel extraction (QIAquickGelExt...

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Abstract

The invention relates to a construction method and application for stably expressing an HPV16 E5 protein cell strain. According to the construction method and application, an HPV16 E5 gene which is mediated by slow virus and provided with an His label is integrated into a Balb / c3T3 cell, and stable expression of the HPV16 E5 cancer gene is achieved. The cell strain can provide a stable cell platform for research of HPV 16 E5 protein biological characteristics and a pathogenic mechanism, and meanwhile a foundation is laid for prevention and control work of HPV-related tumors.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a construction method and application of a cell line stably expressing HPV16E5 protein. Background technique [0002] Lentivirus vector is a gene therapy vector developed on the basis of type I human immunodeficiency virus. Different from general retroviral vectors, lentiviral vectors can effectively integrate foreign genes into host chromosomes to achieve persistent expression. The lentiviral expression vector contains the genetic information required for packaging, transfection, and stable integration. It is necessary to use the expression vector and the packaging plasmid to co-transfect the cells at the same time, and to package the virus in the cells. The packaged virus particles are secreted into the extracellular medium, and after centrifugation to obtain the supernatant, high-titer virus particles are produced, which can be directly used for host cell infection. Af...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC07K14/005C12N15/86C12N2710/20022C12N2740/15043
Inventor 钟儒刚李凡李劲涛汪杨俊琦
Owner BEIJING UNIV OF TECH
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