基于serpin重组蛋白的豆状囊尾蚴病间接ELISA诊断方法的初步建立  

Development of indirect ELISA for the diagnosis of Cysticercus pisiformis infection based on recombinant serpin

作　　者：王莉杰 张少华[1] 毛立[1] 梁盼红 王力[1] 王立群 骆学农[1] WANG Li-jie, ZHANG Shao-hua, MAO Li, LIANG Pan-hong, WANG Li, WANG Li-qun, LUO Xue-nong(State Key Lab oratory of Ve te rinary Etiologic al Biology/Key Lab oratory of Veterinary Public He alth, Minis try of Agriculture~Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences ,Lanzhou 730046, Chin)

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农业部兽医公共卫生重点开放实验室甘肃省动物寄生虫病重点实验室

出　　处：《中国兽医科学》2018年第4期443-449,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31772726,31372433)

摘　　要：为检测兔豆状囊尾蚴（Cysticercus pisiformis）感染，利用cDNA末端快速扩增技术（RACE）扩增豆状囊尾蚴serpin基因，并对其开放阅读框（ORF）及编码的氨基酸序列进行生物信息学分析。构建pET-30a（＋）-Cpserpin重组表达载体，并在大肠杆菌BL21（DE3）中诱导表达。以纯化的重组蛋白（rCpserpin）为包被抗原．优化反应条件，建立检测兔豆状囊尾蚴病血清抗体的间接ELISA方法。结果表明，Cpserpin基因的完整ORF长为l149bp，含有serpin家族保守基序（DEEGAE）、serpin标签序列（FKVDHPFIFFI）以及serpin特有的反应中心环结构域（RCL）；rCpserpin主要以包涵体形式表达，且能被家兔豆状囊尾蚴阳性血清所识别。所建立的间接ELISA的最佳反应条件包括：rCpserpin包被质量浓度为2μg／mL，待检血清和酶标二抗的稀释度分别为1：100和1：2500，包被液和封闭液分别为pH9．6碳酸盐缓冲液和10g/L牛血清白蛋白。利用上述条件检测60份家兔豆状囊尾蚴阳性血清和108份阴性血清，证实该方法的临界值为0．368，敏感性和特异性分别为96．7％和93．5％。结果表明，基于Cpserpin重组蛋白的间接ELISA方法可用于家兔豆状囊尾蚴病的血清学诊断。To detect the infection of Cysticercus pisiformis,the Cpserpin gene was amplified by rapid amplification of cDNA ends （RACE）,and its 0RF and the encoded amino acid sequence were analyzed by bioinformatics, pET-30a （＋）-Cpserpin recombinant expression vector was constructed and induced in Escherichia coli BL21（DE3）.An indirect ELISA for the detection of antibodies against C. pisiformis was developed by optimizing conditions using purified recombinant protein（rCpserpin） as coating anti- gen. The results showed that the Cpserpin gene 0RF was 1 149 bp in length,and had a serpin motif（DEEGiE）, serpin signature （FKVDHPFIFFI） and a specific reactive center loop motif（RCL）,rCpserpin was expressed as inclusion bodies,and could be recognized by C. pisiformis positive serum. The optimal reaction conditions of the indirect ELISA were as follows：the coated mass concentration of rCpserpin was 2 μg/mL,the tested serum dilution and the second antibody dilution respectively were 1：100 and 1：2 500,and the co- ating buffer and the blocking buffer respectively were pU9.6 CBS and 10 g/L bovine serum albumin. By detecting 60 positive serum and 108 negative serum samples using the optimal reaction conditions,the results confirmed that the cut-off value was 0.368,and the sensitivity and specificity respectively were 96.7%and 93.5%.The results suggested that this indirect ELISA based on rCpserpin can be used for serological diagnosis of cysticercosis pisiformis in rabbit.

关 键 词：豆状囊尾蚴 Cpserpin 原核表达 间接ELISA 

分 类 号：S852.734[农业科学—基础兽医学]