The Construction Of Small Hairpin RNA Lentiviral Expression Vector And Its Effect On The Expression Of Human SR-PSOX Gene

Objectives SR-PSOX/CXCL16 is a recently discovered type of chemokine in the recent years, which is related closely with the development of atherosclerosis, but its function is not still explicit. To construct the lentiviral expression vector containing shRNA which could silence human SR-PSOX gene expression, in order to establish a platform of researching human SR-PSOX gene function by RNA interfering technique.Methods In the gene function's research domain, RNAi can produce special, highly effective and fast silence effect to the targeting specific gene, it has been widely applied in the mammal gene function research. In the study, the coding region of human SR-PSOX gene was analysed through the professional Invitrogen software, and the appropriate single-stranded oligonucleotide of shRNA ( small hairpin RNA) targeting human SR-PSOX gene was designed and synthetized. After annealing to generate double-stranded oligo-nucleotides(ds oligo),the ds oligo was cloned into the pENTR?/U6 entry vector which containing RNA Polymeraseâ…¢express element by T4 DNA ligase. The pENTRTM/U6 entry vector containing shRNA which targeting human SR-PSOX gene was constructed by gene cloning and then the homologus shRNA lentiviral expression vector was constructed using LR recombinant technique after identifying the entry vector by DNA sequencing methods. We obtained proper recon with PCR identification and packaged the shRNA lentiviral expression vector into viral host 293FT cells by using liposome-mediated transfection, and 72 hours later we collected the supernatant including false viral particle to infect HepG2 cell which can express human SR-PSOX gene in vitro. The expression of human SR-PSOX gene was detected by using RT-PCR ,indirect immunofluorescence and western-blotting. Results The shRNA lentiviral expression vector targeting human SR-PSOX gene was constructed successfully. The expression levels of mRNA and protein of human SR-PSOX gene was significantly suppressed by the expression vector, the inhibition ratio could reach 80%. And it can induce the silence of human SR-PSOX gene efficiently in cultured cells.Conclusions The shRNA lentiviral expression vector targeting human SR-PSOX gene was constructed successfully. The expression of human SR-PSOX gene was efficiently suppressed by the lentiviral expression vector in cultured cells, thus lay the foundation for studying the function of the gene using RNAi technology.