牛乳腺炎无乳链球菌表面蛋白pgk抗原优势区的原核表达及其间接ELISA检测方法的建立  

Prokaryotic expression of pgk gene of bovine Mastitis Streptococcus agalactiae and establishment of indirect ELISA

作　　者：布日额[1,2] 任晓峰[3] 吴金花[1,2] 王学理[2,4] 刘燕[1,2] 锡林高娃[1,2] 孙立杰[1,2] 刘洋[2,4] 

机构地区：[1]内蒙古民族大学生命科学学院,内蒙古通辽028043 [2]内蒙古民族大学乳源性致病菌研究所,内蒙古通辽028043 [3]东北农业大学动物医学院,黑龙江哈尔滨150000 [4]内蒙古民族大学动物科技学院,内蒙古通辽028043

出　　处：《中国预防兽医学报》2013年第4期312-316,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31060352);第45批中国博士后科学研究基金(20090451027);内蒙古自治区科技厅科技创新引导计划项目(20111802)

摘　　要：为评价牛乳腺炎无乳链球菌重组表面蛋白(pgk)免疫原性,并通过建立检测抗体的间接ELISA方法对免疫抗体效价进行评价,本研究根据GenBank登录的无乳链球菌pgk基因序列,设计一对引物,以临床分离菌株基因组DNA为模板PCR扩增出pgk蛋白抗原优势区的编码基因序列。将其插入pET-30a(+)中,并在大肠杆菌中表达。Western blot鉴定表明,表达的重组蛋白与阳性血清具有良好的反应原性。采用纯化的表达产物作为包被抗原建立了检测无乳链球菌抗体的间接ELISA方法,确定抗原最佳包被浓度为3.115μg/mL,血清最佳稀释度为1∶160,酶标二抗最适稀释度为1∶4 000。初步应用于检测无乳链球菌、化脓性链球菌、大肠杆菌、金黄色葡萄球菌小鼠阳性血清的检测。结果表明牛乳腺炎无乳链球菌pgk亚单位抗原具有良好的抗原性,抗体检测灵敏度达到1∶4 000,同时表现良好的特异性。To express the pgk gene of bovine Streptococcus agalactiae and establish indirect ELISA assay for the antibody detection, the antigenic epitope sequence of pgk gene was amplified by PCR from clinical isolate with a pair of primers derived from the published sequence. The pgk gene sequence was cloned into prokaryotic expression vector pET-30a（＋） for expression in bacteria BL21 and its immunogenicity was confirmed by western blot analysis. An indirect ELISA assay was established using purified expression product as coating antigen for detection of antibody against S.agalactiae. Preliminary application in detecting positive serum against S.agalactiae, S.pyogenes, E.coli and S.aureus showed that this method had high specificity and sensitivity, which was applicable in clinical diagnosis.

关 键 词：无乳链球菌 pgk亚单位 免疫效果 

分 类 号：S852.5[农业科学—基础兽医学]