致猪水肿病大肠杆菌标准菌株F107/86 fedA基因的克隆与原核表达

Cloning and prokaryotic expression of fedA gene of Escherichia coli standard strain F107/86 causing edema disease in pigs

以致猪水肿病大肠杆菌标准菌株F107/86的基因组DNA为模板,对去掉信号肽的456bp的fe-dA基因进行了PCR扩增,将其克隆到原核表达载体pGEX-6P-1的BamHⅠ与EcoRⅠ位点之间,经酶切鉴定与测序,在大肠杆菌中以IPTG诱导表达,并对表达产物用SDS-PAGE和Western-blot分析进行验证。结果表明,筛选出的阳性质粒pGEX-fedA-2(pfedA)经测序并与GenBank中收录的M61713序列进行比较,证明插入序列及读码框正确;表达产物GST-FedA经SDS-PAGE分析大小约42.4ku,Western-blot分析证实其具有良好的反应原性。所构建的原核表达载体可大量表达抗原蛋白,为进一步研究猪水肿病亚单位疫苗奠定了基础。

The fedA gene（456 bp） in absent of its signal sequence was amplified by PCR from Escherichia coli stabdard strain F107/86 which causes edema disease in pigs,and cloned into the prokaryotic expression vector pGEX-6P-1 between the sites of BamHⅠ and EcoRⅠ.The recombinant plasmid was expressed in the E.coli BL21（DE3） induced by IPTG,and the expression product was identified by SDS-PAGE and Western-blot analyses.The intervening sequence of recombinant plasmid pGEX-fedA-2（pfedA） and its reading frame were in the right size by sequencing and comparing with M61713 available in the GenBank.The expression product GST-FedA was 42.4 ku,and showed good reactinogenicity in Western-blot analysis.The results indicated that the fusion proteins were successfully expressed.The present study provided a fundamental basis for further studying on subunit vaccine for prevention of edema disease.