轮状病毒VP4蛋白与肠产毒性大肠杆菌热稳定肠毒素蛋白的融合表达

Fused gene construction and expression of RV VP4 and ST of enteotoxigenic Escherichia coli

利用DNA重组技术将轮状病毒 (RotavirusRV)VP4蛋白主要抗原编码区基因与产肠毒素大肠杆菌 (Enteotoxi genicEscherichiacoliETEC)热稳定肠毒素 (Heat -stableenterotoxinEscherichiacoliST)基因融合 ,插入原核表达质粒 pET -2 8a(+)中 ,经PCR、酶切、测序分析表明已将vp4 -st片段克隆入PET - 2 8a(+) ,而且具有正确的读码框和方向性 ,正确构建了VP4 -ST的融合表达载体 pET2 8a -VP4 -ST。表达质粒转化受体菌BL2 1(DE3 ) plysS ,取经IPTG诱导后表达的目的蛋白进行SDS -PAGE、凝胶薄层扫描分析。结果表明 :表达的目的蛋白以包涵体的形式存在 ,大小约 4 0 2kDa,表达的蛋白量占菌体总蛋白的 13 0 4 8%。

The fused gene of RV VP4 and ST of enteotoxigenic Escherichia coli was inserted into expression plasmid vector pET 28a(+) by DNA recombination technology By PCR,RE digestion and sequence analysis the fused gene VP4 ST in plasmid vector pET 28a(+) had a correct coding frame and orientation This suggested that the recombinant plasmid pET28a VP4 ST was constructed The recombinant plasmid was transformed into E coli BL 21 (DE 3 )plysS The expressed fusion protein VP4 ST was valued by SDS PAGE with expected molecular size of 40 2 kD and the expression efficiency was estimated at 13 048%