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高等学校化学研究 ›› 2011, Vol. 27 ›› Issue (4): 623-627.

• Articles • 上一篇    下一篇

Construction of B7x Gene Overexpression Lentiviral-based Vector

SUN Mei1, JIANG Rui2, SUN Li-hua3, LI Jin-dong4, WANG Bin4, JIN Cheng-yan4 and ZHANG Xing-yi4*   

  1. 1. Department of Pathology, the Second Hospital of Jilin University, Changchun 130041, P. R. China;
    2. Department of Orthopedic Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, P. R. China;
    3. Department of Anesthesia, the Second Hospital of Jilin University, Changchun 130041, P. R. China;
    4. Department of Thoracic Surgery, the Second Hospital of Jilin University, Changchun 130041, P. R. China
  • 收稿日期:2010-11-29 修回日期:2011-03-16 出版日期:2011-07-25 发布日期:2011-06-29
  • 通讯作者: ZHANG Xing-yi E-mail:xyzhang@jlu.edu.cn
  • 基金资助:

    Supported by the National Natural Science Foundation of China(No.30870354), the Fund of Jilin Provincial Science and Technology Services, China(Nos.20070720, 200805120, 20090732) and the Jilin Provincial Development and Reformation Committee Fund, China(No.2009Y042J12314).

Construction of B7x Gene Overexpression Lentiviral-based Vector

SUN Mei1, JIANG Rui2, SUN Li-hua3, LI Jin-dong4, WANG Bin4, JIN Cheng-yan4 and ZHANG Xing-yi4*   

  1. 1. Department of Pathology, the Second Hospital of Jilin University, Changchun 130041, P. R. China;
    2. Department of Orthopedic Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, P. R. China;
    3. Department of Anesthesia, the Second Hospital of Jilin University, Changchun 130041, P. R. China;
    4. Department of Thoracic Surgery, the Second Hospital of Jilin University, Changchun 130041, P. R. China
  • Received:2010-11-29 Revised:2011-03-16 Online:2011-07-25 Published:2011-06-29
  • Contact: ZHANG Xing-yi E-mail:xyzhang@jlu.edu.cn
  • Supported by:

    Supported by the National Natural Science Foundation of China(No.30870354), the Fund of Jilin Provincial Science and Technology Services, China(Nos.20070720, 200805120, 20090732) and the Jilin Provincial Development and Reformation Committee Fund, China(No.2009Y042J12314).

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被引次数

4

摘要： To construct overexpression lentiviral-based vector carrying rat B7x gene, B7x gene precursor sequences amplified by polymerse chain reaction(PCR) were ligated with pLVTHM to generate pLVTHM-B7x gene expression lentiviral-based vector. The positive clones were selected to be submitted to DNA sequencing. HEK293T cells were co-transfected with pLVTHM-B7x and two packaging plasmids psPAX2 and pMD2G to produce lentivirus which express B7x gene. The mRNA expression levels of B7x gene and virus titer were detected by real-time PCR in HEK293T cells. The B7x protein levels were detected by Western blot analysis in HEK293T cells. The identification of restriction enzyme digestion and DNA sequencing confirmed that pLVTHM-B7x lentiviral-based vector was successfully constructed. Green fluorescence was observed in HEK293T packaging cells by means of an inverted fluorescence microscope and the virus titer measured was 2×108 TU/mL. It will establish the foundation for studing deeply the biological function of B7x to construct successfully B7x expression lentiviral-based vector.

关键词: B7x gene, Lentivirus, Overexpression

Abstract: To construct overexpression lentiviral-based vector carrying rat B7x gene, B7x gene precursor sequences amplified by polymerse chain reaction(PCR) were ligated with pLVTHM to generate pLVTHM-B7x gene expression lentiviral-based vector. The positive clones were selected to be submitted to DNA sequencing. HEK293T cells were co-transfected with pLVTHM-B7x and two packaging plasmids psPAX2 and pMD2G to produce lentivirus which express B7x gene. The mRNA expression levels of B7x gene and virus titer were detected by real-time PCR in HEK293T cells. The B7x protein levels were detected by Western blot analysis in HEK293T cells. The identification of restriction enzyme digestion and DNA sequencing confirmed that pLVTHM-B7x lentiviral-based vector was successfully constructed. Green fluorescence was observed in HEK293T packaging cells by means of an inverted fluorescence microscope and the virus titer measured was 2×108 TU/mL. It will establish the foundation for studing deeply the biological function of B7x to construct successfully B7x expression lentiviral-based vector.

Key words: B7x gene, Lentivirus, Overexpression