狂犬病病毒HEP-Flury株N基因的修饰及原核表达  

Modification and Prokaryotic Expression of Nucleoprotein Gene of Rabies Virus Strain HEP-Flury

作　　者：张良[1] 黄永亮[1] 官培英[1] 王怡飞[1] 徐晓娟[1] 吴晓薇[1] 郭霄峰[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《华南农业大学学报》2012年第1期102-105,共4页Journal of South China Agricultural University

基　　金：国家自然科学基金(30871876);公益性行业(农业)科研专项(201103032)

摘　　要：为了高效表达狂犬病病毒HEP-Flury的核蛋白,选取抗原决定簇富集区,利用生物信息学技术分析了狂犬病病毒HEP-Flury N蛋白的核苷酸序列.根据大肠埃希菌Escherichia coli对密码子的偏嗜性,首先对所选取的核苷酸序列进行修饰、引入酶切位点,然后定向克隆至原核表达载体pET-32a(+),构建重组质粒pET-32-NP.将其转化表达宿主菌Escherichia coli BL21(DE3)pLysS,以IPTG诱导表达.表达产物经SDS-PAGE电泳,发现在相对分子质量34 000处有1条特异的蛋白带,与预期大小一致.Western-blotting检测结果显示,该蛋白可被鼠抗His单克隆抗体及犬抗RV多克隆抗体特异识别,表明所表达的N蛋白具有良好的免疫原性.Using bioinformatics analysis, a rich antigenic determinant area in HEP-Flury nucleoprotein se- quence was selected, and the codons were modified to cater the eodon usage bias in Escherichia coli, and the restriction enzyme sequences were inserted in upstream and downstream. The modified gene was syn- thesized and cloned into prokaryotic expression vector pET-32a（ ＋ ）. The recombinant plasimd was des- ignated pET-32-NP, pET-32-NP was175 transformed to E. coli BL2! （DE3）pLysS, and the recombinant protein was expressed after IPTG induction. SDS-PAGE analysis showed that the expressed protein had a relative molecular mass of 34 000. Western-blotting results also revealed that the expressed protein could be recognized specifically by mouse anti-His monoclonal antibody and mouse anti-RV polyclonal antibody.

关 键 词：狂犬病病毒 核蛋白 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]