产气荚膜梭菌β_(1)毒素蛋白的原核表达及其间接ELISA方法的建立  

Prokaryotic expression of β_(1) toxin protein of Clostridium perfringens and establishment of indirect ELISA method

作　　者：张广林 徐龙[1,2] 高云艳 李玲霞 刘永生 尚佑军[1] 曹小安[1] ZHANG Guang-lin;XU Long;GAO Yun-yan;LI Ling-xia;LIU Yong-sheng;SHANG You-jun;CAO Xiao-an(Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences/State Key Laboratory of Pathogenic Biology of Livestock Diseases,Lanzhou 730046,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《中国兽医科学》2021年第10期1207-1214,共8页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2018YFDO5OO9OO);国家现代农业产业技术体系项目(NYCYTX-39)。

摘　　要：为建立产气荚膜梭菌β_(1)毒素抗体间接ELISA检测方法,从C型产气荚膜梭菌扩增出编码β_(1)毒素蛋白的基因,将其克隆至原核表达载体pET30a后,转入表达宿主菌BL21中,用IPTG诱导表达的包涵体蛋白经Ni柱纯化后,用不同浓度的尿素溶液进行复性处理。用不同浓度的重组蛋白包被酶标板,控制变量法对ELISA各步反应条件和血清、酶标二抗的稀释度进行优化。结果表明,纯化后的重组蛋白的纯度可达93%,且具有较好的反应原性,重组蛋白的最适包被浓度为1μg/m L、4℃过夜包被,山羊血清按照1∶300稀释,37℃反应15 min,家兔抗山羊酶标抗体按照1∶50000稀释,37℃反应60 min时效果最好。对30份阴性血清的检测结果进行统计学分析,确定其临界判定值,即D_(450)≥0.3357为阳性,D_(450)<0.3357为阴性;并用相应的血清检验了该方法的敏感性、特异性和重复性。用此方法检测西北地区部分羊场的500份血清样品,其中有473份β_(1)毒素抗体呈阳性,检出率为87.4%。此方法的建立为产气荚膜梭菌β_(1)毒素抗体检测试剂盒的研发奠定了基础。The aim of this study was established an indirect ELISA method for detecting β_(1) toxin antibody of Clostridium perfringens.The gene encoding β_(1) toxin protein was amplified from Clostridium perfringens type C and cloned into prokaryotic expression p ET-30 a vector.The inclusion protein induced by ITPG was purified by Ni column and renatured with different concentrations of urea solution. Different concentrations of recombinant protein were coated with enzyme-labeled plate,and then reaction conditions of ELISA were optimized by control variable method. The experimental results show that the purity of the recombinant protein can reach 93% and has good reactivity. The optimum coating concentration of the recombinant protein is 1 μg/m L and overnight coating at 4 ℃.The best conditions were that sheep serum is diluted 1 ∶300 times at 37 ℃ for 15 min and rabbit anti-sheep enzyme second antibody is diluted at 37 ℃ for 1∶50 000 times. The results of 30 negative sera were statistically analyzed for determining the critical value,that is,D_(450)≥0.335 7 was positive and D_(450)<0.335 7 was negative.The sensitivity,specificity and repeatability of ELISA were testified by detecting to corresponding sera.This method was used to detect 500 serum samples from some sheep farms in Northwest China,and the result showed that 473 samples were positive for β_(1) toxin antibody,with a detection rate of 87.4%.This method laid a foundation for the development of antibody detection kit for β_(1) toxin of Clostridium perfringens.

关 键 词：产气荚膜梭菌 β_(1)毒素蛋白 ELISA 

分 类 号：S852.616.3[农业科学—基础兽医学]