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目的:克隆人类疱疹病毒8型(human herpesvirus 8,HHV-8)ORF50截短基因和编码病毒IL-6(vIL-6)全长基因,将其置于大肠杆菌中作融合表达,并纯化融合表达的vIL-6。方法:分别以本实验室先前构建的重组质粒pcDNA3.1+ORF50和pcD-NA3.1+vIL-6为模板,PCR扩增目的基因片段,克隆到原核表达载体pGEX-6p-1中,构建含HHV-8ORF50截短基因的重组原核表达质粒pORF50-C1,pORF50-C2,pORF50-C3和含vIL-6全长基因的重组原核表达质粒pvIL-6。重组质粒经酶切鉴定和核酸序列测定正确后转化大肠杆菌(E.coli)BL21(DE3)感受态细胞,以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白。用glutathione Sepharose 4B亲和层析柱纯化vIL-6表达产物。用SDS-聚丙烯酰胺凝胶电泳(PAGE)和Western blot检测GST融合蛋白的表达和纯化情况。结果:核酸序列分析的结果表明,克隆的序列与基因数据库中已登记的HHV-8相应序列呈现100%同源;IPTG诱导后的菌体,经SDS-PAGE,显示有相应大小的融合表达蛋白产生。Western blot亦在预期的位置检测到特异性条带。结论:在E.coli中获得了正确表达的HHV-8ORF50基因3个截短片段和vIL-6全长基因,并成功纯化了vIL-6融合蛋白。
OBJECTIVE: To clone ORF50 truncated gene of human herpesvirus 8 (HHV-8) and encode the full-length gene of virus IL-6 (vIL-6), put it into E. coli for fusion expression and purify the fusion The expressed vIL-6. Methods: The recombinant plasmid pcDNA3.1 + ORF50 and pcD-NA3.1 + vIL-6 previously constructed in our laboratory were used as templates to amplify the target gene fragment and cloned into the prokaryotic expression vector pGEX-6p-1 respectively The recombinant prokaryotic expression plasmids pORF50-C1, pORF50-C2 and pORF50-C3 containing the HHV-8 ORF50 truncated gene and the recombinant prokaryotic expression plasmid pvIL-6 containing the full-length vIL-6 gene were obtained. The recombinant plasmids were identified by restriction enzyme digestion and sequenced. The recombinant plasmids were transformed into E.coli BL21 (DE3) competent cells and induced by isopropylthio-β-D-galactoside (IPTG). The vIL-6 expression product was purified by glutathione Sepharose 4B affinity chromatography. The expression and purification of GST fusion protein were detected by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot. Results: The results of nucleic acid sequence analysis showed that the sequence of cloned HHV-8 was 100% homologous with the corresponding sequence of HHV-8 registered in the gene database. After IPTG induction, the correspondingly sized fusion protein was expressed by SDS-PAGE produce. Western blot also detected specific bands at the expected positions. CONCLUSION: Three truncated fragments of HHV-8 ORF50 gene and the full-length vIL-6 gene were obtained in E. coli and the vIL-6 fusion protein was successfully purified.