基于胞内劳森菌重组flgE蛋白间接ELISA检测方法的建立及应用  

Establishment and application of an indirect ELISA based on recombinant protein flgE of Lawsonia intracellularis

作　　者：廖荣莉 郑金 熊梦霞 罗灵芝 赵墩[1,2] 周阳 唐红剑 葛猛 余兴龙[1] LIAO Rong-li;ZHENG Jin;XIONG Meng-xia;LUO Ling-zhi;ZHAO Dun;ZHOU Yang;TANG Hong-jian;GE Meng;YU Xing-long(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Combetter Biotechnology Co.,Ltd.,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]湖南康保特生物科技有限公司,湖南长沙410128

出　　处：《中国预防兽医学报》2021年第2期150-157,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(31972713)。

摘　　要：为建立以胞内劳森菌(LI)重组鞭毛钩基体复合蛋白flgE为包被抗原的间接ELISA检测方法,本研究将flgE基因克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET28a-flgE,转化至大肠杆菌BL21(DE3)感受态细胞,经诱导后获得了可溶性表达的flgE重组蛋白。以纯化的重组flgE蛋白为包被抗原,经优化各反应条件后建立了检测LI抗体的间接ELISA方法。特异性试验结果显示所建立的ELISA方法与CSFV、PRV、PRRSV、PCV2、FMDV、Mhp和APP等阳性血清均无交叉反应。该方法能检测到1600倍稀释的LI阳性血清,批内和批间变异系数均小于10%。利用该方法检测多个不同类型猪场的猪血清(血清来源:2个种猪场1~5胎次的母猪;3个商品猪场30日龄~140日龄的猪;仔猪30头,从30日龄跟踪至80日龄,共采集4次血清),结果显示,母猪和育肥猪的感染率较高可达100%;母源抗体下降后,自50日龄~70日龄起到育肥猪阶段,LI阳性率和抗体水平随着日龄的增长而不断上升;30头跟踪仔猪在单独密闭的饲养条件下,从开始到全群感染LI所需的时间较短(35 d左右)。这些结果与目前已报道的LI血清流行病学调查研究一致,表明该方法能较准确反映LI感染猪的抗体水平。上述结果表明本研究建立的间接ELISA抗体检测方法,具有良好的特异性、敏感性和重复性,可初步用于临床LI血清流行病学的调查。To develop an indirect ELISA for detection of antibody to Lawsonia intracellularis(LI)using recombinant flagellar hook-basal body complex protein flgE as antigen,the flgE gene was cloned into prokaryotic expression pET-28a(+)vector,resulting in recombinant expression plasmid pET-28a-flgE.The pET-28a-flgE was transformed into E.coil strain BL21(DE3)and the soluble recombinant protein flgE was obtained after induced.The purified recombinant protein flgE was used as the coating antigen to develop an indirect ELISA assay for detecting LI antibody after optimization of reaction conditions.The specificity tests showed that the method had no cross reactions with other positive sera,such as CSFV,PRV,PRRSV,PCV2,FMDV,Mhp and APP.It can detect the LI positive serum with the maximum dilution of 1600.In addition,the coefficients of variations in both intra-and interassay were less than 10%.This method was used to detect the serum from several different types of piggery(these serum included:sows of 1 to 5 parities from 2 breeding farms;Pigs of different weights aged from 30 to 140 days from 3 commercial farms;Thirty piglets were followed from 30 to 80 days of age,and serum was collected for 4 times during this period).The results showed that the infection rate of sows and finishing pigs was as high as 100%.After the maternal antibody decreased,the positive rate and antibody level increased with the increase of age during 50-70 days of age to finishing pigs.The time from the initial LI infection to the whole group infection was relatively short(about 35 days)when 30 tracking piglets were reared in isolation.These results are consistent with reported studies so far and showed that this method could accurately reflect the antibody level and antibody positive rate of LI infected pigs.These results indicated that the indirect ELISA antibody detection method established in this study had strong specificity,high sensitivity and good repeatability.It can be preliminarily used for clinical seroepidemiological survey of LI.

关 键 词：猪增生性肠病 胞内劳森菌 鞭毛蛋白flgE 间接ELISA 

分 类 号：S852.61[农业科学—基础兽医学]