牛坏死杆菌43kDa外膜蛋白真核质粒在BHK细胞中的瞬时表达

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。

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

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.