牛坏死杆菌43kDa外膜蛋白真核质粒在BHK细胞中的瞬时表达  

Eukaryotic expression of 43 kDa OMP gene of bovine Fusobacterium necrophorum in BHK cells

作　　者：张思瑶[1] 王志慧[1] 贺显晶[1] 蒋剑成 汪锋锋 王丽娜 肖佳薇 郭东华[1] ZHANG Si-yao;WANG Zhi-hui;HE Xian-jing;JIANG Jian-cheng;WANG Feng-feng;WANG Li-na;XIAO Jia-wei;GUO Dong-hua(College of A nimal Science and Technology,Helongjiang Bayi Agricultural University Daqing 163319,Heilongjiang Province,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国生物制品学杂志》2020年第2期149-151,156,共4页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2017YFD0502200);国家自然科学基金项目面上项目(31572534);黑龙江八一农垦大学校内培育课题(XA2016-03);黑龙江八一农垦大学研究生创新科研项目(YJSCX2017-Y24)。

摘　　要：目的构建含有牛坏死杆菌43 kDa外膜蛋白(outer membrane protein,OMP)基因的真核表达重组质粒pCAGGS-43 kDa OMP,并检测其在BHK细胞中的瞬时表达。方法将43 kDa OMP核苷酸全基因序列与pCAGGS-HA载体连接,构建重组质粒pCAGGS-43 kDa OMP,转化至大肠埃希菌,将鉴定正确的阳性菌转染BHK细胞,采用间接免疫荧光和Western blot法检测重组蛋白的表达。结果经双酶切及PCR鉴定,重组质粒pCAGGS-43 kDa OMP构建正确;转染pCAGGS-43 kDa OMP的BHK细胞镜下观察可见绿色荧光,Western blot检测可见相对分子质量为43 000的目的蛋白。结论成功构建了pCAGGS-43 kDa OMP真核表达质粒,可在BHK细胞中瞬时表达,为进一步研究牛坏死杆菌感染机制奠定了基础。Objective To construct a eukaryotic expression vector pCAGGS-43 kDa OMP for the 43 kDa outer membrane protein(OMP) of bovine Fusobacterium necrophorum and transiently express in BHK cells. Methods The complete gene sequence of 43 kDa OMP was inserted into the vector pCAGGS-HA. The constructed recombinant plasmid pCAGGS-43 kDa OMP was transformed to BHK cells. The positive clones were transfected to BHK cells,and the expression of recombinant protein was determined by IFA and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pCAGGS-43 kDa OMP was constructed correctly. Green fluorescence was observed in BHK cells transfected with recombinant plasmid pCAGGS-43 kDa OMP. Western blot showed that the target protein,with a relative molecular mass of 43 000,was expressed. Conclusion Recombinant plasmid pCAGGS-43 kDa OMP was successfully constructed and expressed transiently in BHK cells,which laid a foundation of further research on the mechanism of bovine F. necrophorum infection.

关 键 词：牛坏死杆菌 外膜蛋白 BHK细胞 瞬时表达 

分 类 号：S852.615[农业科学—基础兽医学]