人类疱疹病毒8型ORF50基因截短片段和vIL-6全长基因的克隆、表达及表达产物的纯化

Cloning and expression of ORF50 truncated genes and the full length vIL-6 gene of human herpesvirus 8(HHV-8) in E.coli and purifying the vIL-6/GST fusion protein

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 and express the ORF50 truncated genes and the full length vIL-6 gene of human herpesvirus 8 （HHV-8） in E.coli, and purify vIL-6/GST fusion protein. Methods：Three pairs of PCR primers were designed according to 3＇ends of ORF50 gene encoding C-ternlinal amino acids（aa 525-691, aa 513-660, aa 541-691 ）, and a pair of PCR primers was also designed according to the full length vIL-6 gene. The DNA of plasmid pcDNA3.1＋ORF50 or pcDNA3.1 ＋vIL-6 was taken as a template, and the genes were amplified using PCR. Subsequently, amplified gene fragments were digested with the restriction enzymes and then cloned into pGEX-6p-1 with proper reading frame to create recombinant prokaryotic expression plasmids designated as pORF50-C1, pORF50-C2,pORF50-C3 and pvIL-6. After identification with enzyme digestion and nucleotide sequences analysis, the recombinant prokaryotic expression plasmids were transformed into host BL21 （DE3） cells. The GST fusion protein expression was induced with isopropyl-β-D-thiogalactopyranoside（IPTG）. The vIL-6 fusion protein was purified by chromatography Glutathione Sepharose 4B. The expression and the purification of the fusion proteins were detected by SDS-PAGE and Western blot. Results：The isolated sequences were 100% homology with the aimed genes registered in GenBank. The fusion proteins could be detected by SDS-PAGE and Western blot as expected. Conclusion：The ORFSO truncated genes and the full length vIL-6 gene were correctly expressed in E.coli BL21 （DE3）cells. The vlL-6/GST fusion protein was successfully purified.