猪致病性大肠杆菌O9菌蜕的制备及免疫保护力

通过PCR方法克隆噬菌体PhiXl74的融菌基因E,将其与温控表达载体pBV220连接,成功构建温控融菌表达盒,设计扩增温控表达盒的2对引物,使其5′端分别与lacZ基因敲除基因的首尾50bp基因同源,使用Red系统同源重组,使用麦康凯平板筛选、PCR鉴定白色菌落,温控诱导融菌蛋白的表达,制备大肠杆菌O9的菌影,并对其融菌效率及免疫保护力进行了初步研究。结果显示,成功构建了温控融菌表达盒,经同源重组成功获得猪致病性大肠杆菌O9温控融菌株,温控表达溶菌蛋白能够成功抑制细菌的增殖,并使活菌数下降3个指数,对小鼠安全性好,皮下免疫组攻毒保护率达到71.43%。

Preparation and immune efficacy of swine pathogenic E. coli 09 ghost

The lytic gene obtained from phage PhiX174 by PCR was inserted into the pBV220 vector to build the temperature-induced lytic cassette. Two pairs of primers were designed and synthetized according to the published sequences of swine pathogenic E. coli 09 lacZ gene and temperature-induced lytic cassette to amplify functional cassette flanked with homology arms. Red/ET re- combination was brought in to creat the swine pathogenic E. coli 09 ghost and studied its lytic efficiency and protective immunity. The results showed that the temperature-induced lytic cassette and swine pathogenic E. coli 09 temperature-induced lytic strain were successfully constructed. The safety of swine pathogenic E. coli 09 ghosts injected into the subcutaneous of Kunming mice showed that the swine pathogenic E. coli 09 ghost had relative protection rate up to 71.43%.