坏死梭杆菌白细胞毒素基因SH片段的克隆与表达

坏死梭杆菌白细胞毒素是坏死杆菌病的主要致病因子,白细胞毒素基因（lkt）是其编码基因。以分离到的国内牛源坏死梭杆菌FN（A）菌株F4基因组DNA为模板,应用PCR方法扩增白细胞毒素基因SH片段,克隆至pMD18-T载体上,以BamHⅠ和HindⅢ酶切的目的片段SH与相应酶切的pET32a载体连接构建pET32a-SH重组表达质粒,经转化E coli BL21（DE3）后用IPTG进行蛋白诱导,SDS-PAGE检测重组蛋白表达情况。结果表明：扩增基因序列大小为1800bp,SDS-PAGE检测重组蛋白有效表达,表达得到大小为80.2kDa的目的蛋白,采用镍柱亲和层析方法纯化SH重组蛋白,获得了纯度达95%的重组蛋白;经West-ern-blot证实,该蛋白对抗坏死杆菌阳性血清具有反应活性。

Cloning and Expression of the Leukotoxin Gene SH from Fusobacterium necrophorum

The leukotoxin of Fusobacterium necrophorum （FN） is considered to be one of the main virulence factors. The lkt gene encodes for FN. In this study, the SH fragment of lkt gene was amplified by PCR using the F4 genome as the template, which was isolated from the Chinese Fusobacterium necrophorum strain. The fragment was then cloned to the pMD18-T vector for sequencing. Thereafter, the SH fragment was subcloned into the multiple cloning sites of the pET32 to construct pET32a-SH recombinant plasmid, which was then trans-formed into E.coli BL21 （DE3） with IPTG induction for expression. SDS-PAGE was used to analyze the recombinant protein. The results showed that the SH fragment of about 1800 bp was amplified and was about 80.2 kDa. The fusion protein was purified by Ni-NTA affinity chromatography under denature conditions, and their purity was above 95%. Western-blOt analysis indicated the SH fragment had antigenicity against Fusobacterium necrophorum.