蜡样芽孢杆菌溶血素BL的表达及多克隆抗体的制备

The expression of Bacillus cereus hemolysin BL and its preparation of polyclonal antibody

为了获得蜡样芽孢杆菌溶血素BL的三个亚基HblA、HblC和HblD的蛋白质和多克隆抗体,试验采用PCR方法分别扩增得到HblA、HblC和HblD基因片段,基于原核表达载体pET-28a构建重组表达质粒,利用大肠杆菌BL21(DE3)感受态细胞分别表达重组蛋白HblA、HblC和HblD,通过Ni2+亲和层析柱对其进行纯化,用纯化蛋白免疫Balb/c小鼠制备相应的多克隆抗体,通过Western-blot检测蜡样芽孢杆菌BC12培养上清液中的溶血素BL。结果表明:试验成功构建出HblA、HblC和HblD基因的重组表达质粒;获得与预期大小相符的重组蛋白HblA、HblC和HblD,蛋白质条带大小分别为44.7,52.1,46.8 ku;制备出HblC和HblD的多克隆抗体,该抗体均能特异性识别蜡样芽孢杆菌培养上清液中的溶血素BL亚基。说明试验制备的多克隆抗体可用于蜡样芽孢杆菌溶血素BL的检测。

In order to obtain the proteins and polyclonal antibodies of the three subunits HblA,HblC and HblD of Bacillus cereus hemolysin BL,the PCR method was used in this experiment to amplify the HblA,HblC and HblD gene fragments for the construction of recombinant expression plasmids based on the prokaryotic expression vector pET-28 a. E. coli BL21 competent cells were used to express HblA,HblC and HblD respectively, and the prokaryotic expression proteins were purified by Ni2+ agarose column;Balb/c mice were immunized with the purified proteins for the preparation of the corresponding polyclonal antibody;hemolysin BL in culture supernatant of Bacillus cereus BC12 was detected by western-blot. The results showed that the recombinant expression plasmids of HblA,HblC and HblD genes were successfully constructed;the recombinant proteins HblA,HblC and HblD of the same size as expected were obtained, which were 44.7,52.1,46.8 ku, respectively;polyclonal antibodies of HblC and HblD were prepared, and both HblC and HblD polyclonal antibodies could specifically recognize the BL subunit of hemolysin in the culture supernatant of Bacillus cereus. The results suggested that the polyclonal antibody prepared in the experiment could be used for the detection of Bacillus cereus hemolysin BL.