猪链球菌2型溶菌酶释放蛋白部分片段的原核表达及其抗体间接ELISA检测方法的建立  

Prokaryotic expression of a muramidase-released protein (MRP) gene fragment of Streptococcus suis type 2 and development of indirect-ELISA for MRP antibody detection

作　　者：周俊明[1] 何孔旺[1] 倪艳秀[1] 俞正玉[1] 茅爱华[1] 吕立新[1] 温立斌[1] 张雪寒[1] 郭容利[1] 李彬[1] 王小敏[1] 

机构地区：[1]江苏省农业科学院兽医研究所农业部动物疫病诊断与免疫重点开放实验室国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《江苏农业学报》2012年第1期80-85,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31072155);江苏省自然科学基金重点项目(BK2010068);江苏省自然科学基金面上项目(BK2009327);江苏省农业科技自主创新探索性研究项目[CX(11)4040];国家"973"项目(2006CB504403)

摘　　要：为了建立猪链球菌2型溶菌酶释放蛋白(MRP)抗体间接ELISA检测方法,该研究首先克隆了MRP编码基因的部分片段,构建了重组表达质粒pET28a-mrp,并将该质粒转化至大肠杆菌BL21(DE3)进行表达,获得了58 000的重组蛋白,经Ni柱纯化后,免疫印迹显示MRP重组蛋白能够被猪链球菌2型抗体特异性识别。以纯化的MRP重组蛋白作为包被抗原,建立了猪链球菌2型MRP抗体检测间接ELISA方法。该ELISA方法的包被抗原浓度为10μg/ml,待检血清1∶50稀释,检测临床48份未免疫猪链球菌2型疫苗健康猪的血清。当效价(S/P)≥0.22时,判定待检血清样品为阳性;当S/P<0.22时,判定待检血清样品为阴性。运用建立的方法能特异性识别猪链球菌2型抗体,人工感染猪的MRP抗体于攻毒后14～28 d出现峰值,同时提示临床健康猪群的免疫功能存在个体差异。The study focused on muramidase-released protein（MRP） of Streptococcus suis type 2,including the expression of MRP gene in vitro and the establishment of indirect ELISA for detection of Streptococcus suis type 2 antibody based on the recombinant protein.The fragment gene of mrp was amplified using SS2-ZY05719 genome DNA as template and the amplified fragment was inserted into expression vector pET-28a（＋）.The recombinant fragment was verified by restriction endonuclease analysis and nucleotide sequencing,then was transformed into Escherichia coli strain BL21（DE3）.Recombinant protein MRP with molecular weight of 58 000 was obtained by IPTG inducing.The purified recombinant protein MRP was proved to be immunoreactive by Western blotting.An MRP-ELISA diagnostic method was developed with the purified MRP recombinant protein as coated antigen.The optimized concentration of coated antigen was 10 μg/ml and the optimized dilution of sera samples was 1︰50.When S/P≥0.22,the serum sample was determined to be positive.When S/P0.22,the sample was negative.Six pigs were artificially infected by Streptococcus suis type 2 ZY05719 and the MRP antibody level reached peak in 14-28d.The MRP antibody production profile varied in individuals,suggesting that there are differences in the immune function of clinically healthy pigs.

关 键 词：猪链球菌2型 MRP 原核表达 间接ELISA 

分 类 号：S634.3[农业科学—蔬菜学]