Construction of GPx6 Over-Expression Lentiviral Vector and Stably Transfected Cell Line
In order to establish a eukaryotic expression cell line stably expressing glutathione peroxidase6(GPx6),the study carried out gene cloning,construction of lentiviral expression vector,and establishment of expression CHO-K1 cell line with porcine GPx6 gene.PCR amplification primers were designed by Primer5 according to the cDNA sequence of porcine GPx6 gene in the NCBI database.6His-GPx6 was cloned from porcine epididymis,a lentiviral over-expression vector containing 6His-GPx6 was constructed,the three-plasmid packaging system was used for lentiviral packaging,and the lentivirus infected CHO-K1 cells were further screened to obtain positive monoclonal cell lines.The expression effect of GPx6 was determined by immunofluorescence,western blotting and real-time fluorescence quantification.Results showed that GPx6 gene was successfully cloned,a lentivirus over-expression vector containing 6His-GPx6 was constructed,and CHO-K1 cell line containing GPx6 was successfully obtained by infected with lentiviral particles.GPx6 over-expression lentiviral vector was successfully constructed,CHO-K1 cell line expressing the target protein was established,which can provide new ideas for further studies in the improvement of porcine fertility and the application of semen diluent additives.
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