戊型肝炎病毒结构蛋白p166在家蚕细胞及蛹体中的表达  

Expression of hepatitis E virus structural protein p166 gene fragment in BmN cell and silkworm pupa

作　　者：杨瑞丽[1] 王文兵[2] 孟继鸿[1] 

机构地区：[1]东南大学医学院病原生物学和免疫学系,南京210009 [2]江苏大学生命科学研究院,江苏镇江212013

出　　处：《免疫学杂志》2005年第4期288-291,共4页Immunological Journal

基　　金：江苏省自然科学基金资助项目(BK2002053)

摘　　要：目的探讨戊型肝炎病毒结构蛋白基因片段p166(HEVp166)在家蚕/BmNPV表达载体系统中的表达水平。方法应用PCR、基因克隆及共转染等技术将p166插入到家蚕杆状病毒多角体启动子的下游,构建重组病毒;并在家蚕细胞及家蚕蛹中表达p166。用间接免疫荧光试验检测感染36h后家蚕培养细胞中p166蛋白的表达情况。收集感染后144h的蛹样品进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodiumdodecylsulphate-Polyacrylamidegelelectrophoreses,SDS-PAGE)及Westernblot-ting分析。结果获得含有HEVp166基因片段的重组杆状病毒表达载体Bm-HEV166。Bm-HEV166能在家蚕细胞中表达p166蛋白,感染后的培养细胞中出现绿色球状体;感染Bm-HEV166的家蚕蛹血淋巴能高效表达p166蛋白,蛹血淋巴能与抗p166单克隆抗体发生特异性反应,在Mr为19000大小的位置出现一条明显的杂交条带。结论HEVp166基因片段在家蚕蛹中能获得高效表达,为进一步研制HEV口服疫苗奠定基础。Objective To express hepatitis E virus structural protein p166 (HEVp166) in Silkworm/Bombyx mori nucleopolyhedrovirus (BmNPV) expression vector system. Methods Gene fragment of HEVp166 was amplified by polymerase chain reaction (PCR) and then inserted into the downstream of polyhedron promoter of the BmNPV genome. Recombinant virus was constructed by gene cloning, co-transfection, and plaque purification. Then the HEVp166 was expressed in BmN cells and silkworm pupae. The expression of HEVp166 in BmN cell (harvested at 36 h postinfection) was detected by indirect immunofluorescence assay (IIFA), and the expression of HEVp166 in silkworm pupa (harvested at 144 h postinfection) was detected by SDS-PAGE and western blotting. Results The recombinant baculovirus expression vector Bm-HEV166 containing HEVp166 was constructed successfully. The p166 protein was expressed in BmN cells and detected as green globoids by IIFA. The p166 protein was efficiently expressed in silkworm pupae and obtained from pupal hemolymph of the pupae infected with the recombinant virus Bm-HEV166. The expressed protein combined specifically with monoclonal antibody of anti-p166 and formed a protein band of about 19 kD in the western blotting profiles. Conclusion The HEV p166 could be expressed at higher level in silkworm pupae with Silkworm/Bm-(NPV) expression vector system, and might be used as a candidate for studying oral vaccine of HEV.

关 键 词：戊型肝炎病毒 家蚕/BmNPV表达载体系统 表达 间接免疫荧光 

分 类 号：R346[医药卫生—基础医学]