中华蜜蜂囊状幼虫病毒北京分离株VP1蛋白基因序列特征及原核表达  

Molecular characterization and prokaryotic expression of VP1 gene from Beijing isolates of Chinese sacbrood virus

作　　者：李薇[1] 黄家兴 Abebe Jenberie WUBIE 薛菲[1] 国占宝[1] 周婷[1] 徐书法[1] 

机构地区：[1]中国农业科学院蜜蜂研究所、农业部授粉昆虫生物学重点实验室,北京100093

出　　处：《昆虫学报》2014年第4期495-502,共8页Acta Entomologica Sinica

基　　金：国家科技支撑计划课题(2011BAD33B04);农业部“948”计划项目(2012-Z6);国家自然科学基金项目(31072093)

摘　　要：【目的】研究中华蜜蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)VP1蛋白的分子进化特征及遗传多样性。【方法】利用RT-PCR方法,克隆了8株CSBV北京分离株VP1蛋白的基因编码区。【结果】序列分析表明,VP1蛋白基因编码区开放阅读框长945 bp,编码315个氨基酸,推测编码蛋白的相对分子量和等电点分别为35.42 kDa和9.23,具有亲水性和免疫原性。序列同源性分析表明,不同年份CSBV北京分离株VP1蛋白氨基酸序列间差异较小,仅个别氨基酸存在差异。北京分离株与辽宁分离株及越南分离株VP1核苷酸序列一致性达93%,与印度及韩国分离株VP1核苷酸序列一致性达92%,与英国分离株VP1核苷酸序列一致性最低,为88%。序列分析同时表明,CSBV北京分离株VP1蛋白序列存在特有的序列特征,同其他地区分离株比较,北京分离株VP1蛋白序列中存在着氨基酸的插入突变。序列替换率分析表明,亚洲型分离株间序列替换率低于亚洲分离株与欧洲分离株间的替换率。构建原核表达载体pEASY-E1-VP1,经IPTG诱导,CSBV VP1蛋白在大肠杆菌Escherichia coli BL21(DE3)pLysS菌株中表达。【结论】本研究提示CSBV不同分离株基因序列存在变异,结果为进一步研究CSBV致病性分化的分子机理奠定了基础。[ Aim ] To understand the molecular evolution characteristics and genetic diversity of VP1 protein of Chinese sacbrood virus （CSBV） in Apis cerana cerana. [ Methods ] Eight eDNA sequences encoding VP1 proteins were cloned by RT-PCR from Beijing isolates of Chinese sacbrood virus. [ Results] Sequence analysis results showed that the open reading frame （ORF） of VPI gene is 945 bp in length, encoding 315 amino acids with the predicted molecular weight of 35.42 kDa and the theoretical isoeleetrie point of 9.23, and the encoded protein has hydrophilieity and immunogenicity characteristics. Multiple sequence alignment indicated that the VP1 proteins from these CSBV Beijing isolates in different years share high amino acid sequence identity with occasional changes. isolates have high nucleotide sequence identity with those of Liaoning The VP1 genes from Beijing （LN） and Vietnam isolates （93%） , India and Korea isolates （92%）, while have the lowest nucleotide sequence identity with that of UK isolate （88%）. The sequence analysis results also showed that the VP1 gene from Beijing isolates, other than from some isolates in other areas, has a specific amino acid characteristic which includes amino acid insertion. The nucleotide substitution rate among all Asian isolates was less than that between Asian and European isolates. A recombinant plasmid pEASY-E1-VP1, containing the coding sequence of VP1 protein, was constructed using pEASY-E1 as a fused expression vector, and VP1 protein was expressed successfully after induced with isopropyl-beta-D-thiogalactopyranoside （IPTG） in BL21 （DE3） pLysS strain of Escherichia coli. [ Conclusion ] The results provide the basis for further studying themolecular mechanisms of the pathogenicity evolution of CSBV by confirming the existence of mutants in CSBV of different isolates.

关 键 词：中华蜜蜂 囊状幼虫病毒 VP1蛋白 系统进化 原核表达 

分 类 号：Q965.8[生物学—昆虫学]