Cloning Of Human LIGHT Gene And Expression Of The Cloned Molecule On HepG₂ Cells

Objective To analyze LIGHT gene expression in peripheral blood mononuclear cells (PBMCs),to clone LIGHT gene,to construct human LIGHT expressing vector, transform into E.Coli, and transfect human liver cancer cells. Methods PBMCs were conventionally isolated and cultured in RPMI1640,containing 10% FCS. Total RNA was extracted from PBMCs . LIGHT gene expression in PBMCs was analyzed by RT-PCR.The full length LIGHT cDNA was amplified by RT-PCR. The result and the vector pcDNA4C were respectively digested by enzymes BamHI and EcoRI. LIGHT cDNA was connected with the plasmid. Calcium Chloride method was used to make competence. Then the recombinant plasmid was transformed into E.Coli JM109. Culture human liver cancer cell line HepG₂ till logarithmic growth phase. EGFP was transfected into HepG₂ cellsJts expression was observed at 12h,24h and 48h after transfection. LIGHT recombinant plasmid was transfected into HepG₂ cells by liposomes.The expression of LIGHT was detected with ELISA.A standard curve was generated for the set of samples assayed.The statistical significance was tested by SPSS.The concentration of LIGHT protein of 12h,24h,48h,72h,5d after transfection was elicited. Results A 723bp cDNA was amplified by RT-PCR. LIGHT expression in PBMCs could be detected in the group stimulated by PHA. After confirmed by restriction endonulease analysis and connected with pcDNA4C, LIGHT cDNA was successfully transformed into E.Coli JM109. Purified recombinant plasmid was obtained.LIGHT gene sequence was analyzed.Though one base occurred synonymous mutation,it did not change coding amino acid, human liver cancer cell line HepG₂ was cultured and proliferated. After the transfection of EGFP, the recombinant plasmid pcDNA4C-LIGHT could be transfected into HepG₂ cells with 0.4μg DNA,3.2μ1 Enhancer,10μ1 Effectene. The expression of LIGHT was successfully detected with ELISA. The coefficient of correlation was equal to 0.997 and had evident statistical significance.The quantity of LIGHT protein was the greatest at 24h and the least at 5d. Conclusion LIGHT cDNA was successfully cloned and the recombinant plasmid pcDNA4C -LIGHT was constructed. LIGHT was transfected into human liver cancer cells and its expression was successfully detected.