麻疹病毒Edmonston疫苗株血凝素基因克隆及表达

Cloning of hemagglutinin gene of Edmonston vaccine strain of measles virus and fusion protein expression in Escherichia coli

目的 构建麻疹病毒(MV)血凝素蛋白(H蛋白)基因的重组质粒,使其在大肠埃希菌中大量表达H蛋白,为进一步研究奠定基础。方法 从MVEdmonston株减毒活疫苗中提取基因组RNA ,RT PCR扩增H蛋白基因。用限制性内切酶Bam HI和Hind Ⅲ双酶切H基因片段和pGEMEX 1载体,连接后获得重组质粒MV H -pGEMEX 1。然后,用IPTG诱导重组质粒在大肠埃希菌JM10 9(DE3)和BL2 1(DE3)中表达。结果 RT PCR获得的片段长度约为190 0bp ,与MV H基因相符。MV H -pGEMEX 1测序结果显示:MV H基因对码正确,核苷酸序列符合率达99.4 %。转化重组质粒的大肠埃希菌,其表达产物经SDS PAGE蛋白凝胶电泳,出现与目的蛋白分子量相一致的条带。结论 成功构建MV H蛋白表达载体MV H—pGEMEX 1,并在大肠埃希菌中获得表达。

Objective To construct recombinant plasmid MV-H-pGEMEX-1 and express fusion proteins in E.coli in order to lay a foundation for further study on early diagnostic antibody and measles virus (MV) subunit vaccine. Methods MV genomic RNA was extracted from the live attenuated vaccine (Edmonston strain) and H gene was amplified by RT-PCR. After digested by Bam-HI and Hind-Ⅲ enzymes, the H gene was inserted into expression plasmid pGEMEX-1 by T4 DNA ligase, the recombinant plasmid MV-H-pGEMEX-1 was transformed into E.coli BL21 (DE3) and JM109 (DE3) and induced by IPTG. The expression of H protein was analyzed by SDS-PAGE. Results The product of RT-PCR was 1 900 bp which corresponded to MV-H. PCR analysis, enzymatic digestion and DNA sequence analysis verified that the H gene had been inserted into the vector in the correct direction, and sequence analysis showed that compared with the reported H gene published in NCBI, the homology of the sequences was 99.4%, while the amino acid homology was 98.8%. The molecular weight of fusion proteins expressed by E.coli JM109 (DE3) and BL21 (DE3) in SDS-PAGE corresponded to calculated molecular masses. Conclusions Recombinant expression plasmid MV-H-pGEMEX-1 was successfully constructed, which expressed fusion protein in E.coli.