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Cell strain for expressing HER2 gene, and construction method and application thereof

A cell line and gene technology, applied to cells modified by the introduction of foreign genetic material, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problem of limited transplanted tumor models, limited tumorigenesis ability, high-efficiency expression of HER2, etc. question

Inactive Publication Date: 2014-09-03
HEFEI HANKEMAB BIOTECH CO LTD
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Problems solved by technology

However, the number of existing tumor cell lines with high expression of HER2 is limited and limited by the tumorigenic ability, which can be used to establish xenograft tumor models. Therefore, it is necessary to artificially construct tumor cell lines with high efficiency, stable expression of HER2 and good tumorigenic ability
The patent "A HER2 Stable Expression Cell Line and Its Application in Drug Screening" published in 2009 (publication number CN101343633A) transfected the full-length HER2 gene into various host cells with the pEGFPN3 / 1 vector, and screened to obtain stable expression HER2 cell lines, but the problem of how to express HER2 in cells is still unsolved
Because gene amplification takes 6 to 12 months, the protein expression outcome of this method cannot be predicted at an early stage and is the rate-limiting step of the expression technology

Method used

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  • Cell strain for expressing HER2 gene, and construction method and application thereof
  • Cell strain for expressing HER2 gene, and construction method and application thereof
  • Cell strain for expressing HER2 gene, and construction method and application thereof

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Embodiment

[0066] Used material and source thereof are as follows in the embodiment:

[0067] The pBabe / Her2 plasmid was donated by Professor Mark I. Greene of the University of Pennsylvania School of Medicine, and the plasmid containing the full-length Her2 cDNA sequence can also be purchased from OriGene Technologies; the GC-Rich expression vector was purchased from Hangzhou Anpu Company; Lipofetamine2000 was purchased from Invitrogen Company; Her2 antibody Ab-1 was purchased from NeoMarkers.

[0068] 1.1 Her2 expression plasmid construction

[0069] Using the pBabe / Her2 plasmid as a template, design primers to amplify the full-length human Her2 (gene ID2064) gene, digest with EcoRI+NotI, connect with pCDNA3.1 and GC-Rich expression vectors, and transform XL-10 competent Cells, positive clones were selected, and after EcoRI+NotI digestion and sequencing verification, one pCDNA3.1 / Her2 plasmid clone and four GC-Rich / Her2 plasmid clones (numbered 2-3, 2-4, 2 -5 and 2-6). These plasmid...

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Abstract

The invention provides a cell strain for expressing HER2 gene, and a construction method and application thereof. The method comprises the following steps: 1) inserting HER2 gene into the polyclone site of the expression vector to obtain a recombinant expression vector; and 2) transfecting a cell strain by using the recombinant expression vector to obtain the monoclonal cell strain for expressing HER2 gene. The cell strain can efficiently and stably express the HER2 gene, has resistance to the existing HER2 targeted antibody drug trastuzumab, can successfully construct a human breast cancer transplantation tumor model in the nude mouse body, and can be used for screening novel breast cancer drugs.

Description

technical field [0001] The invention relates to a cell line expressing HER2 and its application. Background technique [0002] The HER2 / ErbB2 protein receptor belongs to the epidermal growth factor receptor (EGFR) family, which is a transmembrane protein with receptor tyrosine kinase (RTK) activity encoded by the c-erbB2 (her2 / neu) gene Glycoprotein, with a full-length molecular weight of 185kDa, high expression in malignant tumors such as breast cancer, gastric cancer, and ovarian cancer often indicates that the tumor is prone to metastasis and poor prognosis, and is highly correlated with resistance to radiotherapy, chemotherapy, and endocrine therapy. Because HER2 is expressed on the surface of tumor cells, it is not expressed or only very low expressed in normal adult tissues, making it a good drug target. The first anti-HER2 humanized monoclonal antibody drug trastuzumab (trastuzumab, trade name Herceptin) has shown unique efficacy in the clinical treatment of breast c...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12Q1/02
Inventor 胡思怡查昭沈国栋黄辉刘兢
Owner HEFEI HANKEMAB BIOTECH CO LTD
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