Construction and identification of recombinant human LIGHT lentiviral vector and its expression in human colorectal carcinoma cells

Objective To construct and identify recombinant human LIGHT lentiviral vector pLenti-LIGHT and observe its expression in human colorectal carcinoma cells HCT116.Methods The full length of human LIGHT gene was cloned to lentiviral expression vector by recombinant DNA technology.The positive clones were confirmed by enzyme digestion and DNA sequencing.The recombined lentiviriral particles were produced in 293T cells.The titer of recombinant viruses was tested,and the recombinant viruses were used to transfect into HCT116 cells in different multiple of infection (MOI) by detecting the reporter gene expression.The LIGHT expression was determined by using real-time quantitative polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA).Cellular proliferation inhibitory activity was determined by using methyl thiazolyl tetrazolium (MTT) assay.Results Enzyme digestion and DNA sequencing showed that the LIGHT gene was inserted into the lentiviral vector,correctly.With thetiter of 1.96 × 108 TU/ml,the optimal MOI of the recombined lentivirus for HCT116 was 2 and the transfection efficiency was up to 92％ at the same time.As compared with the control group,the mRNA ratio of LIGHT to GAPDH in LIGHT-transfected group was increased 19.03,68.59,94.35 and 11.71 folds at 24,48,72,120 h respectively after transfection (P ＜ 0.05),respectively.There was no significant difference between lentivirus control group and control group (P ＞ 0.05).In LIGHT-transfected group,the LIGHT protein expression was significantly increased from 0 to 11.36 μg/L at 12 h and 72 h.MTT result revealed the cellular proliferation activity in LIGHT-transfected group was also inhibited.Conclusion The recombinant LIGHT lentivirus expressing vector was successfully constructed.The exogenous gene LIGHT can be expressed efficiently in HCT116 cells and may inhibit the growth of HCT116 cells. Key words: Colorectal carcinoma; Lentivirus; Gene transfection