坏死梭杆菌白细胞毒素基因的BSBSE片段的原核表达及抗血清制备

Expression of leukotoxin BSBSE gene of Fusobacterium necrophorum

本研究以国内分离的牛源坏死梭杆菌F4基因组DNA为模板,应用PCR方法扩增BSBSE片段,克隆到pMD18-T载体上,鉴定并测序正确后,构建pET32a-BSBSE表达质粒,转化E.coli BL21(DE3)经IPTG诱导、SDS-PAGE检测重组蛋白表达、Western blot检测重组蛋白反应原性。以该重组蛋白免疫兔制备抗血清,经间接ELISA检测抗血清效价。结果表明,PCR扩增得到1100bp的BSBSE片段,以此片段构建了pET32a-BSBSE表达质粒,经诱导后获得了目的蛋白表达,以Western blot检测该重组蛋白证明为本研究的目的蛋白,并且与抗体具有反应原性。以间接ELISA检测该重组蛋白免疫兔制备的抗血清效价达105。研究结果将为坏死梭杆菌毒力因子(lkt)的深入研究奠定了物质基础。

BSBSE gene of lkt was amplified by PCR from the F4 genome of bovine origin Fusobacterium necrophorum and cloned to the pET32 to construct pET32a-BSBSE, which was then transformed into E.coli BL21 （DE3） for expression under IPTG induction. SDS-PAGE and Western blot assays showed that the expressed BSBSE had strong immunogenecity against rabbit antiserum. This study would be useful for further investigation of 1kt being a major virulence factor.