维氏气单胞菌TH0426株rpoN蛋白的生物学特性分析及原核表达

Biological characteristics and prokaryotic expression of the rpoN gene of Aeromonas veronii TH0426 Strain

为了深入研究维氏气单胞菌毒力蛋白rpoN的功能,试验采用邻近法构建rpoN基因的系统进化树,对rpoN蛋白进行生物学特性分析,PCR法扩增rpoN基因,将其与表达载体pET-32a(+)连接,再将连接产物转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达和制备抗体,测定抗体效价并进行Western-blot检测。结果表明:rpoN蛋白稳定,属于胞内蛋白,是全α型蛋白。重组表达质粒pET-32a-rpoN在大肠杆菌中成功表达,在约58 ku处可见明亮条带,rpoN多克隆抗体效价为1∶279 000,具有特异性。说明表达的rpoN蛋白具有一定的免疫原性。

In order to further study the function of Aerornonas veronii virulence protein rpoN,the phylogenetic tree of rpoN gene was constructed by proximity method to analyze the biological characteristics of rpoN protein,and rpoN gene sequence was amplified by PCR and linked to pET-32 a(+).The ligation product was then transformed into E.coli BL21(DE3)competent cells for induction of expression and preparation of antibodies.The results showed that the rpoN protein was stable,belonged to the intracellular protein,and it was a full alpha type protein with irregular curl.Recombinant protein pET-32 a(+)-rpon was successfully expressed in Escherichia coli accompanied by bright bands visible at about 58 ku,and rpoN polyclonal antibody was specific.The expression of RpoN protein had certain immunogenicity,which laid a foundation for further research on the relationship between RpoN protein structure,pathogenicity and drug resistance.