摘要
目的筛选与贝氏柯克斯体感染血清反应的贝氏柯克斯体毒力相关蛋白。方法以贝氏柯克斯体标准株九里株为模板,采用PCR扩增包括IV型分泌系统、分泌性蛋白和膜蛋白在内的毒力及毒力相关的基因,将扩得目的基因片段与原核表达质粒pET32a重组,将重组质粒转化大肠杆菌,用贝氏柯克斯体实验感染小鼠血清与转化大肠杆菌作免疫印迹。结果设计173对引物共扩增出170个目的基因,经PCR及酶切鉴定,有155个基因与pET32a质粒重组成功,SDS-PAGE电泳分析发现,共有104个转化菌表达目的蛋白,在104个重组蛋白中,筛选到32个与贝氏柯克斯体感染小鼠血清反应的重组蛋白。结论这些与贝氏柯克斯体感染血清反应的毒力相关蛋白具有良好的免疫原性和免疫反应性。
The total genomie sequence of the international standard strain of Coxiella burnetii RSA 493 was used as template to amplify the virulence and virulence-related genes including those encoding for Ⅳ type secretion system, secretory proteins and membrane proteins with PCR. The amplified target gene fragments were ligated with to prokaryotic expression vector pET32a,and the recombinant plasmid were then used to transform E. coli cells, SDS-PAGE assay was used to identify the expressed recombinant proteins. It was found that 170 target genes had been amplified with 173 pairs of primers designed, in which 155 genes were cloned into plasmid pET32a successively after identification with PCR and endonuclease digestion. As demonstrated by SDS-PAGE,104 transformed bacteria expressed target proteins in which 32 recombinant proteins were recognized by sera of mice infected with C, burnetii. It is evident that these proteins show excellent immunogenicity and immunoreactivity.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2008年第7期593-596,共4页
Chinese Journal of Zoonoses
基金
国家自然科学基金(No.30670101)
国家自然科学青年基金(No.30700744)
关键词
贝氏柯克斯体
毒力
重组蛋白
免疫印迹
Coxiella burnetii
virulence
recombinant protein
immunoblot assay
