禽多杀性巴氏杆菌ompa基因在Vero细胞中的表达

Expression of ompa Gene from Avian Pasteurella multocida in Vero Cells

利用PCR技术扩增出禽多杀性巴氏杆菌1 050 bp的ompa基因片段,克隆到pUCm-T载体上,再亚克隆到真核载体pcDNA3.1(+)上,构建重组质粒pCA(pcDNA3.1-OmpA),将该重组质粒体外转染Vero细胞,检测其转录和表达情况。结果表明重组质粒pCA构建成功,通过RT-PCR由转染了重组质粒的Vero细胞中扩增出了目的条带,间接免疫荧光试验分析表明在转染了pCA的Vero细胞中出现绿色荧光,Western blotting分析显示重组质粒pCA转染的Vero细胞泳道出现约37.5 ku的特异条带,说明ompa基因在Vero细胞中成功表达了活性蛋白,从而为进一步研究禽多杀性巴氏杆菌ompa DNA疫苗的免疫效果奠定了基础。

Avian Pasteu,rella muhocida is the etiological agent of fowl cholera. The OmpA protein is one of the major immunogenie antigen of avian Pasteurella muhocida and plays important roles in inducing immune responses that confer resistance against infections. In the present study,the ompa gene fragment amplified by PCR from avian PasteureUa muhocida was cloned into pUCm-T. Subsequently it was subcloned into the eukaryotic expression vector pcDNA3.1 （＋）, the recombinant plasmid pCA （pcDNA3.1-OmpA）was obtained. Then the recombinant plasmid was transfected into Veto cells in vitro. Transcription and expression of ompa gene was analyzed by RT-PCR,indirect immunofluorescence and Western blotting. The results showed that the recombinant plasmid had been constructed successfully. The target DNA fragment was amplified by RT-PCR from Vero cells transfectcd by recombinant plasmid pCA. Western blotting analysis showed that the recombinant plasmid producted with apparent molecular weight about 37.5 ku. Indirect immunofluorescenee showed that Vero cells transfected by pCA appeared green fluorescence. In conclusion,the target activated protein had been successfully expressed. The research developed the foundation for the ompa DNA vaccine against fowl cholera.