腐败梭菌α毒素和产气荚膜梭菌ε毒素的融合表达纯化及其免疫效力评价  

Fusion expression and purification of Clostridium septicumαtoxin and Clostridium perfringensεtoxin and its evaluation of immune efficacy

作　　者：夏中秋 赵妍 王浩[2] 马国军 姚伦广[1,3] XIA Zhongqiu;ZHAO Yan;WANG Hao;MA Guojun;YAO Lunguang(College of Life Science and Agricultural Engineering,Nanyang Normal University,Nanyang 473061,China;Northwest A&F University,Yangling 712100,China;Henan Provincial Research Center for Livestock and Poultry Health Products Engineering Technology,Nanyang 473061,China)

机构地区：[1]南阳师范学院生命科学与农业工程学院,河南南阳473061 [2]西北农林科技大学,陕西杨凌712100 [3]河南省畜禽保健品工程技术研究中心,河南南阳473061

出　　处：《黑龙江畜牧兽医》2022年第15期115-119,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金面上项目(31870917);河南省高校科技创新团队项目(20IRTSTHN024)。

摘　　要：为了获得腐败梭菌α毒素和产气荚膜梭菌ε毒素融合蛋白并评价其免疫保护效力,试验采用PCR扩增方法将两种毒素融合基因ETX-CSA克隆至pvexk载体中,选取阳性克隆子转化至大肠杆菌BL21(DE3)感受态细胞中,然后使用1%乳糖诱导阳性菌株进行蛋白质表达,通过SP阳离子柱纯化获得融合蛋白,通过SDS-PAGE、Western-blot验证融合蛋白的表达情况,通过将融合蛋白与佐剂混合配制成疫苗免疫家兔获取血清,然后使用免疫血清进行中和试验研究融合蛋白的免疫原性和免疫中和效应。结果表明:成功获得了可溶性表达的融合蛋白ETX-CSA,将纯化的ETX-CSA蛋白与佐剂混合配制成疫苗后免疫家兔所产生的血清对腐败梭菌毒素的中和效价达到1 MLD/0.1 mL(MLD为最小致死量),对D型产气荚膜梭菌毒素的中和效价达到40 MLD/0.1 mL。说明融合蛋白成功表达,其具有免疫原性,免疫兔所产生的血清能够同时中和腐败梭菌毒素和D型产气荚膜梭菌毒素。In order to obtain the fusion protein of Clostridium septicumαtoxin and Clostridium perfringensεtoxin and to evaluate its immunoprotective efficacy,in the experiment,the two-toxin fusion gene ETX-CSA was cloned into the vector pvexk by PCR amplification;positive clone was selected and transformed into E.coli BL21(DE3)competent cells,and then 1%lactose was used to induce protein expression of positive strains;the fusion protein was obtained by cation column purification,and the expression of the fusion protein was verified by SDS-PAGE and Western-blot;the sera were obtained from the rabbits immunized by a vaccine which was made by mixing the fusion protein and adjuvant,and then the immune sera were used to conduct a neutralization test to study the immunogenicity and immune neutralization effect of the fusion protein.The results showed that the soluble expressed fusion protein ETX-CSA was successfully obtained;the sera,produced by rabbits immunized by the vaccine which was made by mixing purified ETX-CSA protein and adjuvant,had a neutralizing titer of 1 MLD/0.1 mL against Clostridium septicum toxin,and reached a neutralizing titer of 40 MLD/0.1 mL against D-type Clostridium perfringens toxin.The results suggested that the fusion protein was successfully expressed and had immunogenicity,and the sera produced by immunized rabbits could neutralize both Clostridium septicum toxin and D-type Clostridium perfringens toxin.

关 键 词：腐败梭菌 产气荚膜梭菌 Α毒素 ε毒素 融合表达 纯化 免疫 

分 类 号：S852.61+6.3[农业科学—基础兽医学] S852.4[农业科学—兽医学]