Construction of PSCSI-GFP lentiviral vector for the endothelin-converting enzyme-like 1 gene

Objective To design and construct the RNA interference (RNAi) lentiviral vector targeting the endothelin-converting enzyme-like 1 (ECEL1) gene. Methods The RNA interference target was designed with the ECEL1 gene as the template, and the short-hairpin RNA (shRNA) interference sequence was designed based on the selected target sequence. Enzyme cut sites for restriction endonuclease were added to both ends of this sequence to synthesize single-stranded DNA oligo, which was then paired in annealing buffer to form double-stranded DNA oligo. GV115 vectors were linearized by double digestion with Age I and EcoR I enzymes, and then they were connected with the DNA oligo; the grafted product transformed Escherichia coli receptor cells and was amplified, sequenced, and identified by PCR.The recombinant ECEL1 gene RNAi lentivirus was obtained by plasmid extraction, transfection, concentration, and purification, and HIV-1 p24 antigen ELISA was used to determine the titer of the recombinant RNAi lentivirus. Human hepatoma BEL-7404 cells were transfected with the qualified lentivirus, and a control group was set up. Fluorescence observation was performed to determine transfection rate. Real-time PCR and Western blot were used to measure the mRNA and protein expression of target gene after ECEL1 gene knockdown in human hepatoma BEL-7404 cells. The two-independent-samples t test was used for comparison between two groups. Results After the design with the ECEL1 gene as the template, the psc48784 fragment was selected as the target for RNAi; the double-stranded DNA oligo was prepared and the positive recons were identified and sequenced by PCR, and the results showed that psc48784 was the correct clone. The ECEL1 gene RNAi lentivirus was successfully constructed after plasmid extraction, transfection, concentration, and purification and was qualified for physical test and sterility test. HIV-1 p24 antigen ELISA showed that the ECEL1 gene RNAi lentivirus had a virus titer of 3 E+ 8 TU/ml, suggesting that the qualified ECEL1 gene RNAi lentivirus with a high titer was successfully constructed. After the human hepatoma BEL-7404 cells were transfected with the ECEL1 gene RNAi lentivirus, fluorescence observation showed a transfection efficiency of80% and a stable state of cells. Real-time PCR and Western blot showed significant reductions in the mRNA and protein expression of the ECEL1 gene in human hepatoma BEL-7404 cells in the experimental group (P < 0. 05) , and the knockdown efficiency reached 70. 5%.Conclusion The ECEL1 gene PSCSI-GFP lentiviral vector is successfully constructed, and stable virus samples with a high titer are obtained. The stable human hepatoma BEL-7404 cells with ECEL1 gene knockdown are also obtained.