Construction and identification of porcine SBP1 gene overexpression and interference recombinant lentiviral vector
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摘要: 【目的】构建硒结合蛋白1基因（SBP1）过表达与干扰重组慢病毒载体，并通过感染猪骨骼肌卫星细胞验证SBP1基因的过表达和沉默效果，为后续揭示SBP1基因调控肌纤维类型转化及猪肉品质的分子机理打下基础。【方法】在对猪SBP1蛋白进行生物信息学分析的基础上，根据GenBank中猪SBP1基因mRNA序列（XM_021089863.1） PCR扩增SBP1基因全长序列，克隆至过表达慢病毒载体pHBLV-CMV上构建SBP1基因过表达重组慢病毒载体；同时针对猪SBP1基因设计3个干扰靶点序列和1个阴性对照序列，分别克隆至干扰慢病毒载体pHBLV-U6上构建SBP1基因干扰重组慢病毒载体。以构建的重组慢病毒载体感染猪骨骼肌卫星细胞，经嘌呤霉素筛选获得稳定表达细胞系，并采用实时荧光定量PCR和Western blotting检测SBP1基因及其蛋白的表达情况。【结果】猪SBP1蛋白分子式为C2523H3916N678O727S24，相对分子量为56.14839 kD，理论等电点（pI）为6.40，为稳定的分泌蛋白；存在39个潜在磷酸化位点，包括20个丝氨酸磷酸化位点、6个酪氨酸磷酸化位点及13个苏氨酸磷酸化位点。以构建的SBP1基因过表达与干扰重组慢病毒载体分别感染猪骨骼肌卫星细胞，经嘌呤霉素筛选2代后，在荧光显微镜下均能观察到绿色荧光。实时荧光定量PCR检测结果表明，过表达组的SBP1基因相对表达量极显著上调32.0倍（P<0.01，下同） ，干扰组中以shRNA-3的沉默效果达极显著水平，对应的SBP1基因相对表达量下调58.45%； Western blotting检测结果显示，过表达组的SBP1蛋白表达量极显著上调，而shRNA-1、shRNA-2和shRNA-3干扰组的SBP1蛋白表达量均极显著下调。【结论】基于猪骨骼肌卫星细胞成功构建了猪SBP1基因过表达稳定表达细胞系，同时鉴定出shRNA-3对SBP1基因的沉默效果最佳并获得稳定沉默细胞系，为后续揭示SBP1基因调控猪肉品质的分子机理打下了基础。Abstract: 【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.
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Keywords:
- pig /
- SBP1 gene /
- skeletal muscle satellite cells /
- overexpression /
- interference /
- recombinant lentiviral vector
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