Construct PSCSI-GFP Lentiviral Vector Of ECEL1 Gene

Objective: To construct a lentiviral vector of ECEL1 gene.Method : 1.According to ECEL1 base as template,RNA interference target is designed.According to the selected target sequence,short hairpin RNA is designed.(sh RNA)The interference sequence was used to synthesize single-stranded DNA oligocytes by adding corresponding restriction endonuclease sites at both ends,and then paired to form double-stranded DNA oligo in annealing buffer.The carrier is linearized by using agei and Eco RI double enzymes to cut GV115 vectors.By connecting the carrier with the DNA oligo,the grafted product transformed Escherichia coli receptor cells and was amplified by PCR and sequenced and identified.The recombinant ECEL1 gene RNAi lentivirus was obtained by plasmid extraction,transfection,concentration and purification.The titer of the recombinant RNAi was determined by "HIV-1 p24 antigen ELISA method".2.After plasmid extraction,transfection,enrichment and purification,the recombinant lentivirus was obtained,and the titer was determined by the "HIV-1 p24 antigen ELISA method".ECEL1 gene RNAi lentivirus infection of human hepatoma cells(BEL-7404)and set up control group,fluorescence observation showed infection rate.Real-time PCR was used to detect the m RNA expression of target gene after knockout in cells.Results:1.After the ECEL1 gene template was designed,the psc48784 fragment was selected as the target of RNA interference,and the double-stranded DNA oligosis-PCR was prepared and sequenced to verify that psc48784 was the correct clone.After the plasmid was extracted,transfected and harvested,the ECEL1 gene RNAi lentivirus was successfully constructed.Physical test,aseptic test and titer test.The virus droplet of ECEL1 gene RNAi slow virus was detected by HIV-1 P24 antigen ELISA,and the titer of samples was 3e+8TU/ml.2.After the transfection of 293 T cells,the lentivirus was packaged and purified,and the high titer of ECEL1 gene RNA was used to disturb the lentivirus.ECEL1 gene RNAi lentivirus infection in human hepatoma cells(BEL-7404),infection efficiency reached more than 80%.The expression of ECEL1 gene at m RNA level was inhibited(p<0.05),and the knock-out efficiency reached 70.5%.Conclusion: Successfully constructed the ECEL1 gene PSCSI-GFP lentiviral vector and obtained the stable high titer virus samples.The stable ECEL1 gene knockout of human liver cancer cells(BEL-7404),which laid the foundation for further discussion of ECEL1 gene on the function of hepatocellular carcinoma cells.