Construction and identification of porcine SBP1 gene overexpression and interference recombinant lentiviral vector

【Objective】 This paper constructed a recombinant lentiviral vector for selenium-binding protein 1 gene (SBP1) overexpression and interference,and verified the overexpression and silencing effect of SBP1 gene by infecting the porcine skeletal muscle satellite cells,to lay the foundation for the subsequent revelation of SBP1 gene regulating the transformation of muscle fiber type and molecular mechanism of pork quality.【Method】 Based on the bioinformatics analysis of porcine SBP1 protein,the full-length sequence of SBP1 gene was amplified by PCR according to the mRNA sequence of porcine SBP1 gene in GenBank(XM_021089863.1),and cloned into the overexpression lentiviral vector pHBLV-CMV to construct the recombinant lentiviral vector of overexpression of the SBP1 gene. At the same time,three interfering target sequences and one negative control sequence were designed for the porcine SBP1 gene and cloned into the interfering lentiviral vector pHBLV-U6 to construct the interfering recombinant lentiviral vector for the SBP1 gene.Porcine skeletal muscle satellite cells were infected with constructed recombinant lentiviral vectors. Stable expression cell lines were obtained by puromycin screening. And the expression of SBP1 gene and its proteins were detected by realtime fluorescence quantitative PCR and Western blotting.【Result】 Porcine SBP1 protein had a molecular formula of C₂₅₂₃H₃₉₁₆N₆₇₈O₇₂₇S₂₄,a relative molecular weight of 56.14839 kD,a theoretical isoelectric point(pI) of 6.40,and was a stable secreted protein;there were 39 potential phosphorylation sites,including 20 serine phosphorylation sites,6 tyrosine phosphorylation sites and 13 threonine phosphorylation sites. Porcine skeletal muscle satellite cells were infected with constructed SBP1 gene overexpression and interference recombinant lentiviral vectors,respectively,and green fluorescence was observed under fluorescence microscope after puromycin screening for 2 generations.The results of real-time fluorescence quantitative PCR showed that the relative expression of SBP1 gene in the overexpression group was extremely significantly up-regulated by 32.0 times(P<0.01,the same below),and the silencing effect of shRNA-3 in the interference group reached extremely significant level,corresponding to the down-regulation of the relative expression of the SBP1 gene by 58.45%. Western blotting showed that the SBP1 protein expression in the overexpression group was extremely significantly up-regulated,while the SBP1 protein expression in the shRNA-1,shRNA-2 and shRNA-3 interference groups were all extremely significantly down-regulated.【Conclusion】 Based on porcine skeletal muscle satellite cells,a stable expression cell line ofporcine SBP1geneis successfully constructed. At the same time,the best silencing effect of shRNA-3 on SBP1 gene is identified and a stable silencing cell line is obtained. The foundation is laid for the subsequent revelation of molecular mechanism of SBP1 gene in regulating the quality of pork.