猪胞内劳森菌抗体间接ELISA检测方法的建立及应用  

Development and application of an indirect ELISA for detection of antibody for porcine Lawsonia intracellularis

作　　者：裴艳艳 张梦琳 许浒 相丽润 张洪亮[1] 彭金美[1] 王倩[1] 田志军[1] 周国辉[1] EI Yan-yan;ZHANG Meng-lin;XU Hu;XIANG Li-run;ZHANG Hong-liang;PENG Jin-mei;WANG Qian;TIAN Zhi-jun;ZHOU Guo-hui(State Key Laboratory for Animal Disease Control and Prevention/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第1期55-60,69,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费课题(1610302022008)。

摘　　要：猪增生性肠病(PPE),通常被称为猪回肠炎(PI),由胞内劳森氏菌(LI)感染引起。为建立检测LI抗体的间接ELISA方法,本研究构建LI外膜蛋白基因(LI0841)的重组表达质粒p ET28a-LI0841,经测序无误后转化BL21(DE3)宿主菌,经IPTG诱导表达,采用SDS-PAGE检测重组蛋白LI0841蛋白(rLI0841)的表达形式,经Ni柱纯化后采用western blot鉴定其反应原性。SDS-PAGE结果显示,在32 ku处出现目的条带,且其主要以可溶性形式表达;western blot结果显示,r LI0841能够与兔LI多克隆抗体特异性反应,表明纯化的r LI0841具有较强的反应原性,可作为包被抗原用于建立间接ELISA检测方法,经各反应条件优化初步建立LI抗体的间接ELISA检测方法。各反应条件的优化结果显示,4.38 ng/孔的r LI0841以4℃过夜包被最佳;血清最佳稀释度为1∶100,37℃反应0.5 h;羊抗猪Ig G-HRP最佳稀释度为1∶10000,37℃作用0.5 h;TMB底物37℃显色15 min。利用建立的间接ELISA方法检测猪繁殖与呼吸障碍综合征病毒、猪伪狂犬病病毒、副猪嗜血杆菌、猪链球菌、传染性胸膜肺炎放线杆菌及经美国Biostone PPE抗体检测试剂盒检测为阳性的猪血清,评估该方法的特异性;将LI阳性血清2倍倍比稀释(1∶100~1∶51200)后,采用本研究建立的间接ELISA方法检测,评估该方法的敏感性;以同一批次和不同批次包被的酶标板分别检测5份不同LI抗体水平的猪血清,评估该方法的重复性。结果显示,该方法除能检测到LI阳性血清外,其余相关病原的阳性血清均为阴性,特异性较强;LI阳性血清1∶800稀释时检测结果仍为阳性,敏感性较高;对5份不同抗体水平的LI阳性血清的批内、批间重复性试验的变异系数均小于10%,重复性较好。利用该ELISA方法与美国Biostone公司PPE抗体检测试剂盒同时检测104份临床猪血清样品,比较二者的检测结果,并计算二者的符合率;采用建立的间接ELISA方法检测黑龙江、吉林等�Porcine proliferative enteropathy(PPE),commonly known as porcine ileitis(PI),is caused by Lawsonia intracellularis(LI)infection.In this study,a recombinant expression plasmid pET28a-LI0841 containing the LI outer membrane protein gene(LI0841)was constructed,sequenced and then transformed into BL21(DE3)host bacteria.IPTG was added to induce expression,and the expression form of recombinant LI0841 protein(rLI0841)was determined by SDS-PAGE.After purification by Ni column,the expressed protein was identified by western blot.The results of SDS-PAGE showed that the target band appeared at 32 ku,and it was mainly expressed in a soluble form.The western blot results showed that rLI0841 could react specifically with the rabbit LI polyclonal antibody,and thus could be used as a coating antigen to establish an indirect ELISA detection method.The optimization results of each reaction condition of the indirect ELISA method as follows:the optimal coating concentration of rLI0841 was 4.38ng/well at 4℃overnight;the best dilution of serum was 1∶100 and the duration of the incubation was 0.5 hour at 37℃;the goat anti-pig IgG-HRP was 1∶10000 dilution and the duration of the incubation was 0.5 hour at 37℃;TMB substrate developed color at 37℃for 15 minutes.The established method could specifically detect LI antibody and had no cross-reactivity with PRRSV,PRV,HPS,SS,APP pig positive serum.The coefficients of variation(CV)of intra-and inter-repeatability tests were both less than 10%,indicating good specificity,sensitivity and repeatability.Compared with AsurDxTM Lawsonia Intracellularis antibody test kit,the compliance rate of the two methods was 91.35%.The established indirect ELISA method was used to detect 413 clinical pig serum samples from Heilongjiang,Jilin and other regions.The positive detection rate of LI was 59.81%(247/413),indicating that LI was prevalent in pig herds in Heilongjiang,Jilin and other regions.This study established a specific ELISA antibody detection method against LI,and provided a method for mo

关 键 词：胞内劳森氏菌 间接ELISA 抗体检测 LI0841重组蛋白 

分 类 号：S852.61[农业科学—基础兽医学]