基于PLD蛋白的伪结核棒状杆菌血清抗体间接ELISA检测方法的建立与应用
尹 峥，刘 刚，王晶晶，李晨露，徐海玲，张 琪*，许信刚*
（西北农林科技大学 动物医学院，陕西 杨凌 712100）
摘要：为了建立以PLD蛋白为包被抗原的伪结核棒状杆菌（Corynebacterium pseudotuberculosis，Cp）血清抗体间接ELISA检测方法，以原核表达得到的重组PLD蛋白作为包被抗原，并进行特异性、敏感性和重复性试验，以及对临床样本进行检测。结果显示，重组PLD蛋白大小为33 ku，Western-blot检测具有良好反应原性；建立的间接ELISA检测方法最佳抗原包被量为每孔200 ng，血清以1:200稀释作用1 h，酶标二抗以1:5 000稀释作用1 h，TMB显色20 min，血清阴阳性临界值为0.367。特异性试验表明，建立的ELISA方法对羊布鲁菌、产气荚膜梭菌、羊结核、羊衣原体和小反刍兽疫阳性血清检测结果均为阴性，特异性良好；灵敏性试验表明，当Cp标准阳性血清1:1 280 稀释时检测结果仍为阳性，灵敏性良好；重复性试验表明，批内和批间变异系数分别在2.27%~8.89%和2.11%~6.92%，重复性良好。对临床采集的山羊样本进行检测与实际患病情况的符合率为96%，符合率较高。对陕西省不同羊场的643份山羊血清进行检测，Cp阳性率为31%。本研究建立的Cp抗体检测方法为下一步研制Cp抗体ELISA检测试剂盒提供理论基础。
关键词：伪结核棒状杆菌；PLD；原核表达；间接ELISA；抗体检测
The establishment and application of indirect ELISA detection method for Corynebacterium pseudotuberculosis antibody based on PLD protein
YIN Zheng, LIU Gang, WANG Jing-jing, LI Chen-lu, XU Hai-ling, ZHANG Qi*, XU Xin-gang*
(College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China)
Abstract：In order to establish an indirect ELISA detection method for Corynebacterium pseudotuberculosis (Cp) serum antibodies, the recombinant PLD protein expressed in prokaryotic cells was used as the coating antigen.Specificity, sensitivity and repeatability tests were conducted, and clinical samples were tested by the indirect ELISA. In result,the recombinant PLD protein was 33 ku and Western-blot showed good reactogenicity. The optimal antigen coating volume of the Cp indirect ELISA detection method was 200 ng. The serum was diluted at 200 for one hour, the enzyme-labeled antibody was diluted with 5 000 for one hour, and the TMB for 20 minutes. The critical value of serum is 0.367. Specificity experiments showed that the indirect ELISA detection method has negative results for other positive serum and has good specificity. Sensitivity test showed that when the Cp standard positive serum was diluted 1 280, the test results were still positive and the sensitivity was good. The repeatability test showed that the coefficient of variation within and between batches was from 2.27% to 8.89% and from 2.11% to 6.92%, with good repeatability. The coincidence rate between the detection of clinically goat samples and the actual disease was 96% and the coincidence rate was high.The positive rate was 31% in 643 goat serum samples from different sheep farms in Shaanxi province.In conclusion,the Cp antibody detection method established in this study provides a theoretical basis for the next development of Cp antibody ELISA detection kit.
Key words：Corynebacterium pseudotuberculosis; PLD; prokaryotic expression; indirect ELISA; antibody testing
*Corresponding authors:XU Xin-gang,E-mail：286567031@qq.com;ZHANG Qi,E-mail，273010466@qq.com
以伪结核棒状杆菌（Corynebacterium pseudotuberculosis，Cp）为病原的羊干酪性淋巴结炎 （caseous lymphadenitis, CLA）又称羊伪结核病，可引起浅表淋巴结及肝脏、肺脏等多个脏器的炎症反应，出现大小不等的结节，严重时引发器官的衰竭，造成死亡 [1]。动物感染后，机体会出现营养不良、生产性能下降等症状，给养殖业带来巨大经济损失[2]。目前，针对该病的治疗药物及疫苗产品相对较少，对病灶部位消毒处理是该病的主要治疗手段[3]。因此，对于羊伪结核病的防控以及抗体水平的检测越来越受到重视。间接ELISA方法在抗体检测方面以其成本低、周期短、操作便捷、灵敏度高、适宜大量样本检测等优点而在疾病诊断领域应用广泛并且常常用于质控[4]。
PLD蛋白是Cp的主要毒力因子之一。它是一种外毒素，通过水解动物细胞膜鞘磷脂的酯键而増加血管渗透性，导致血浆从血管渗出进入周围组织，使病原菌能够从嗜中性粒细胞逃逸，并且损害朝向感染部位的嗜中性粒细胞趋化性。PLD是Cp的主要保护性抗原，常被用于防制该病原的疫苗研发[5]。本试验以Cp的PLD蛋白为目标，通过构建PLD蛋白重组表达载体，实现PLD蛋白的高效表达；并建立以重组PLD蛋白作为包被抗原，用于检测Cp抗体水平的间接ELISA检测方法。本试验研究结果为开展Cp的临床检测提供技术支持，为Cp抗体ELISA检测试剂盒的研究奠定基础。
1 材料与方法
1.1 菌种与质粒
Cp陕西分离株由西北农林科技大学动物医学院兽医微生物实验室分离鉴定并保存[6]。pET-28a(+)质粒、pGM-T载体质粒、感受态E.coli DH5α 以及E.coli BL21(DE3)均为天根生化有限公司产品。
1.2 主要试剂
细菌基因组DNA提取试剂盒、DNA Marker、TMB单组份显色液为天根生化科技公司产品。质粒小提试剂盒、Protein Marker、胶回收试剂盒、2×Es Taq MasterMix均为北京全式金生物技术公司产品。BamHⅠ、XhoⅠ限制性核酸内切酶为NEB公司产品。T4 DNA连接酶为TaKaRa公司产品。HRP-Rabbit-Anti-Goat IgG为笛医生物公司产品。酶标板为BEAVER公司产品。Proteinlso Ni-NTA Resin为上海七海复泰生物科技公司产品。BCA蛋白定量试剂盒为 |