胸膜肺炎放线杆菌ApxⅠ、ApxⅢ和ApxⅣ的截短表达及免疫反应性鉴定

Truncated expression and immunoreactivity identification of ApxⅠ,ApxⅢand ApxⅣof Actinobacillus pleuropneumoniae

为了制备胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)亚单位疫苗和建立配套的鉴别诊断方法,在蛋白抗原性和亲疏水性分析的基础上,设计了针对溶血外毒素(Apx)Ⅰ、Ⅲ、Ⅳ截短蛋白的基因扩增引物,以实验室保存的血清2型和血清5型菌株基因组DNA作为模板进行特异性基因片段扩增,分别将目的片段克隆至原核表达载体pColdI后转化入Rosetta感受态细胞,通过1 mmol/L异丙基硫代半乳糖苷(IPTG)诱导表达,应用小鼠抗6×His组氨酸单克隆抗体和猪多抗对表达的重组蛋白上清进行Western blot分析。结果表明:PCR扩增产物与预期大小相符,通过酶切鉴定和测序证明成功构建了重组表达质粒pColdI-tApxⅠ、pColdI-tApxⅢ和pColdI-tApxⅣ。诱导表达的蛋白分子量分别为63、50和54 kDa,大小符合预期,可以稳定表达可溶性蛋白。Western blot显示表达的蛋白具有良好的免疫反应性。该试验的成功开展将为制备APP亚单位疫苗和建立配套的鉴别诊断方法奠定基础。

In order to prepare a subunit vaccine of Actinobacillus pleuropneumoniae(APP)and establish a matching differential diagnosis method,primers for the genes of the ApxⅠ,ApxⅢ,ApxⅣtruncated proteins of Actinobacillus pleuropneumoniae-RTX-toxins(Apx)were designed.The genomic DNA of the serotypes 2 and 5 strains was used as templates for application of specific gene fragments.The target fragments were then cloned into the prokaryotic expression vector pColdI and transformed into Rosetta competent cells.The expression was induced by 1 mmol/L IPTG.The expressed recombinant protein supernatants were identified by the 6×His monoclonal antibody and polyclonal antibodies from pigs using western blot.The results showed that the PCR amplification products were in accordance with the expected sizes,and the recombinant expression plasmids pColdI-tApxⅠ,pCold-tApxⅢ,and pColdI-tApxⅣwere successfully constructed by enzyme digestion identification and sequencing.The recombinant proteins were induced successfully and their molecular weight was 63,50 and 54 kDa,respectively.The recombinant proteins were expressed especially in soluble forms and in a stable manner.Western blot showed that the expressed proteins reacted specifically with the 6×His monoclonal antibody and polyclonal antibodies of swine,which indicated a good immunoreactivity.The successful expression of the three proteins laid a foundation for preparation of a subunit vaccine of APP and establishment of supporting differential diagnosis methods.