摘要
为了提高产气荚膜梭菌β毒素(CPB)亚单位疫苗的免疫效果,试验将不同破伤风毒素辅助性T细胞表位引入该蛋白前,用OEPR(Overlap Extension PCR and Recombination)法构建了8个重组表达质粒(即p Cold-CPB、p Cold-P2-CPB、p Cold-P21-P30-P2-CPB、p Cold-P32-P23-P21-P30-P2-CPB和p ET22b-CPB、p ET22b-P2-CPB、p ET22b-P21-P30-P2-CPB、p ET22bP32-P23-P21-P30-P2-CPB)。将阳性重组质粒转入到大肠杆菌中,以自诱导培养基表达目的蛋白。SDS-PAGE分析结果表明,8个重组菌均成功表达出目的蛋白,且大小均与预期一致。Western Blotting检测结果表明,重组蛋白均可被C型产气荚膜梭菌阳性血清识别。毒性试验结果表明,重组蛋白均可致死小鼠。本研究首次用OEPR法成功构建了8个含不同破伤风毒素T细胞表位的产气荚膜梭菌β毒素表达质粒,并成功表达了目的蛋白,为探索破伤风毒素辅助性T细胞表位对产气荚膜梭菌β毒素免疫效果的影响及进一步开发亚单位疫苗奠定了基础。
In order to improve the immune effect of the β-toxin subunit vaccine of C. perfringens through the introduction of different tetanus toxin T cell epitopes into the protein, eight recombinant expr-essions plasmids（ pCold-CPB,pCold-P2-CPB, pCold-P21-P30- P2-CPB, pCold-P32-P23-P21-P30-P2-CPB andpET22b-CPB, pET22b-P2-CPB, pET22b-P21-P30-P2-CPB, pET22b-P32-P23-P21-P30- P2-CPB） were constru-cted using the OEPR （Overlap Extension PCR and Recombination） method . The positive recombinant plas-mid was transferred into E. coli, and the tin-get protein was expressed in the self inducing medium. The results of SDS-PAGE analysis showed that the 8 recombinant bacteria successfully expressed the target protein, and the size was consistent with the expected molecular weight. Western Blotting results showed that the recombinant protein could be recognized by Clostridium perfringens type C positive serum. The toxicity test results showed that the recombinant protein killed mice. This study was the first to successfully construct 8 expression plasmids of Clostridium peoeringens beta-toxin containing different T-cell epitopes of tetanus using OEPR method, and successfully express the target protein, which can be used to explore the effect of tetanus toxin T cellepitopes on the immune effect of C. perfringens beta toxin and to further develop subunit vaccines.
作者
董令赢
彭小兵
彭国瑞
李旭妮
蒋玉文
DONG Ling-ying ; PENG Xiao-bing ; PENG Guo-rui ; LI Xu-ni ; JIANG Yu-wen(China Institute of Veterinary Drug Control , Beijing 100081, China)
出处
《中国兽医杂志》
CAS
北大核心
2018年第7期33-38,I0004,共7页
Chinese Journal of Veterinary Medicine
基金
国家重点研发计划"牛羊重要细菌病新型疫苗及其工艺研究(2017YFD0500905)"
作者简介
董令赢（1993-），女，硕士生，主要从事兽用细菌生物制品学研究，E—mail：915633864@qq．com;通讯作者：彭小兵，E-mail：6733038882@qq．com.;通讯作者：蒋玉文，E-mail：jiangyuwen@ivdc．org．cn
