Construction of a fusion gene expression vector containing human granulysin and green fluorescent protein gene and its expression in murine macrophage RAW264.7

    YI Zhengª²Jun, ZHU Daoª²Yin, LI Junª²Ming, HE Yongª²Lin, YANG Jian, LI Na

    Department of Microbiology and Immunology, Chongqing University of Medical Science, Chongqing 400016, China

    ¡¾Abstract¡¿ AIM: To clone the human granulysin from activated cytotoxic Tª²lymphocytes (CTL), construct an eukaryotic expression vector containing the fusion gene of granulysin (GLS) and green fluorescent protein reporter and observe its expression in murine macrophage RAW264.7 strain. METHODS: The coding sequence of the granulysin was amplified from the total RNA of human CTL activated by allogenic antigen after reverse transcription by Nestedª²PCR, inserted into pEGFPª²C1 plasmid and then transfected RAW264.7 cells. The expression of the fusion protein and GLS was detected by fluorescence microscope and RTª²PCR. The immunoreactive of the product was confirmed by immunocytochemistry method. RESULTS: The whole coding sequence of GLS was successfully amplified as expected. The accurate open reading frame (ORF) of the fusion protein recon was confirmed by PCR, endonuclease digestion and sequencing. The fusion protein that was immunoreactive was successfully expressed in the targeted cells. CONCLUSION: Human granulysin can be expressed in murine macrophage RAW264.7 strain in a fusion protein pattern and retain its immunoreactivity. ......