中国株兔出血症病毒衣壳蛋白VP60基因在大肠杆菌中的表达及其免疫原性鉴定

Expression and Identification of the Rabbit Hemorrhagic Disease Virus VP60 Gene

应用 RT- PCR技术扩增编码兔出血症病毒 (rabbit hem orrhagic disease virus,RHDV) WX84株衣壳蛋白 VP6 0基因 ,将 PCR产物按相应的阅读框架克隆到表达性载体 p GEX- 6 P- 1中谷胱甘肽转移酶 (GST)基因的下游。将重组质粒转化入大肠杆菌 BL2 1株 ,在 1.0 m mol/ L IPTG和 37℃的条件下诱导 ,VP6 0 - GST基因融合蛋白获了高效表达。经聚丙烯酰胺凝胶电泳和 Western- blotting试验证实所表达的融合蛋白产物相对分子质量与预期的 870 0 0相符。将表达产物经电泳切胶回收目的条带后免疫小鼠 ,所得抗血清应用间接 EL ISA检测 ,与 RHDV病毒粒子呈阳性反应。试验结果表明 ,大肠杆菌中表达的 RHDV VP6

The rabbit hemorrhagic disease virus(RHDV) capsid protein(VP60) gene was amplified by reverse transcript polymerase chain reaction(RT PCR) technigue.The RT PCR product was cloned into the expressing plasmid vector pGEX 6P 1 .The recombinant was verified by restriction endonuclease analysis and nucleotide sequencing.Then it was transformed into E.coli strain BL21 for VP60 expression.The expression of VP60 gene was indentified by SDS PAGE and Western blotting,and a specific protein band of 87 000 was found as a fussion protein with glutathione transferase protein.The specific band of expression was excised from the gel and used to immunize the mice.The antisera was collected from the immunized mice and detected by ELISA in 96 well plates coated with RHDV.The positive results showed that the in vitro expressed protein of VP60 gene as a fusion protein maintains some antigenicity of RHDV.