布鲁菌S2疫苗株免疫牛与牛种、羊种布鲁菌自然感染牛ELISA鉴别诊断方法的建立

为建立区分猪种布鲁菌S2疫苗株接种奶牛与布鲁菌自然感染奶牛，BLAST比对分析羊种、牛种、猪种、犬种、沙林鼠种和绵羊种6种布鲁菌基因序列，发现repA—related基因是猪种布鲁菌与牛种及羊种布鲁菌的差异基因。设计引物PCR扩增获得repA-related基因片段，克隆并原核表达得到了布鲁菌repA—related融合蛋白，以repArelated蛋白建立间接EI．IsA检测方法。用repA—related蛋白间接ELISA检测猪种s2疫苗株接种动物血清为阳性，检测牛种和羊种布鲁菌自然感染动物血清为阴性。repA—related蛋白间接EusA能从试管凝聚实验（SAT）及常规ELIsA检测阳性的奶牛血清样本中，区分出s2疫苗接种牛与牛种布鲁菌感染牛。

Establishment of a novel diagnostic method for discriminating cattles vaccinated with Brucella suis strain 2 from those naturally infected with Brucella abortus

To develop a method that can discriminate dairy cows inoculated with Brucella suis vac cine strain 2 from those with natural Brucella infection,we compared Brucella gene sequences in six Brucella species including B. rnelitensis, B. abortus, 13. suis, B. canis, B. neotornae, and 13. ovis using BLAST alignment software and found that repA-related gene can discriminate B. suis from 13. suis and B. abortus. PCR amplification primers were designed to obtain the repA-related gene fragments, and repA-related fusion protein in Brucella was harvested through cloning and prokary- otic expression,with an attempt to develop an indirect ELISA method with the repA-related pro tein,which showed that the serum repA-related protein was positive in animals inoculated with 13rucella suis vaccine strain $2 but negative in those with the natural infection of B. abortus or B. rnelitensis. Thus, the repA-related protein-based indirect ELISA could distinguish dairy cows inoc ulated with B. suis vaccine strain S2 from those with the natural infection of B. abortus,whose ser um samples are positive in both serum agglutination test or conventional ELISA.