牛坏死杆菌43ku外膜蛋白截短片段的原核表达

Prokaryotic Expression of the Truncated 43K OMP Genes of Bovine Fusobacterium necrophorum

据GenBank发表的牛坏死杆菌H05菌株43ku外膜蛋白(43KOMP)基因的核苷酸序列,设计了4对含有BamHⅠ和XhoⅠ酶切位点的特异性引物,通过PCR扩增坏死杆菌43KOMP基因相互重叠的4个截短片段,PCR扩增产物经BamHⅠ和XhoⅠ酶切消化后克隆至pET-32a原核表达载体,4个截短片段的阳性质粒转化大肠杆菌BL21(DE3)感受态细胞,通过PCR筛选阳性克隆,然后利用IPTG进行诱导表达。结果显示,牛坏死杆菌43KOMP基因4个截短片段均成功获得了表达,表达的重组融合蛋白大小均为30ku左右,为进一步进行43KOMP蛋白的免疫原性和相关功能研究奠定了基础。

Four pairs of primers containing BamH I and Xho I sites were designed for amplification of 43 ku outer membrane protein （43K OMP） of bovine Fusobacterium necrophorum strain H05 according to the GenBank. The PCR products of the truncated 43K OMP genes were digested with BamH I and Xho I restriction endonuclease, and then the digested products were ligated to the pET-32a vector with His tag. The positive plasmids of four truncated 43K OMP genes were transformed in to E. coli BL21（DE3）. Protein expression of four truncated 43K OMP genes were induced using 1.0 mmol/L IPTG. The result indicated that the four truncated 43K OMP genes were successfully expressed in E. coli, and molecular weights of the expressed proteins were all about 30 ku. This study would provide some basis for further research of immunogenicity of the outer membrane protein of Fusobacterium necrophorurn.