摘要
目的建立猪链球菌2型(SS2)胞外因子(EF)的免疫学检测方法。方法将SS2四川人源分离株ZYH24EF抗原性强的区域进行克隆、原核表达,对表达的融合蛋白(rEF-GST)采用亲和层析法纯化、经凝血酶作用去除标签蛋白获得纯化的EF目的蛋白。以纯化的EF免疫BALB/c小鼠,间接ELISA方法筛选阳性克隆,对获得的抗EF单克隆抗体(McAbs)进行特异性分析,并建立猪链球菌EF检测方法。结果得到rEF-GST和EF相对分子量分别为62000和35000;建立了4株稳定分泌抗EFMcAbs的杂交瘤细胞系2G1、5H7、3H4和1F2;腹水的特异性鉴定结果表明4株McAbs能特异性的识别EF;以2G1单抗腹水和抗EF多克隆抗血清建立的夹心ELISA可特异地检测EF。结论4株抗EFMcAbs的制备对猪链球菌检测试剂盒的研制以及该病的防治有重要意义。
In order to establish an immunological method of detecting the extracellular factor （EF）from Streptococcus suis type 2 （SS2）, the epf gene of SS2 of human origin strain ZYH24 isolated in Sichuan province was truncated and cloned to prokaryotic expression vector pGEX4T-2 and the expressed fusion protein rEF-GST with molecular mass （Mr） of 62 000 was expressed in E. coll. After purification with affinity chromatography and treatment with thrombin protease to cut off GST from EF-GST, thus obtaining a purified EF with Mr 35 000, which showed strong reaction to SS2 positive sera as demonstrated by Weatern blotting. This protein was used as antigen to immunize BALB/c mice intraperitoneally and an indirect ELISA assay coated with this purified EF was used to screen hybridoma cells secreting specific monoclonal antibodies. In these ways, 4 strains of cell lines stably secreting EF-specific monpcloanl antibodies were obtained; they were 2G1, 5H7, 3H4 and 1F2. As demonstrated by the examination of the mouse ascetic fluids, all the monoclonal antibodies produced by these 4 strains of cell lines could recognize EF specifically. The sandwich ELISA assay established by using the 2G1 monoclonal ascetic fluid and the polyclonal anti-EF antibody as the first and second antibodies could be used successively to detect the presence of EF. This method of testing may be value for the development of diagnostic kit as well as for the prevention and treatment of SS2 infection.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2009年第1期44-48,共5页
Chinese Journal of Zoonoses
基金
国家“十五”科技攻关(2003BA712A03-05)
国家自然科学基金(30600533)
江苏省自然基金(BK2008066、BK2006014)
关键词
猪链球菌2型
胞外因子
表达
单克隆抗体
Streptococcus suis 2
extracdlular factor
expression
monoclonal antibody
