山羊副流感病毒3型N蛋白原核表达及间接ELISA抗体检测方法的建立

发布时间：2018-03-12 14:35

本文选题：山羊副流感病毒型　切入点：N蛋白　出处：《畜牧兽医学报》2017年01期 　论文类型：期刊论文

【摘要】：山羊副流感病毒3型可引起山羊呼吸道疾病,与支原体、细菌等混合感染引起广泛的发病与死亡,笔者拟通过建立基于N蛋白的间接ELISA抗体检测方法用于该病的监测。设计引物扩增目的基因N,克隆到质粒pET32a(+)上,构建重组质粒pET32a-N,并转化至大肠杆菌BL21中,IPTG诱导表达重组N蛋白并鉴定。通过条件优化建立ELISA抗体检测方法,并检测临床样品与HI试验进行比较。本试验成功构建重组质粒,诱导表达的蛋白质(47ku)经SDS-PAGE与Western blot鉴定,证明其正确表达且具有良好的抗原性与特异性。利用纯化的蛋白质作为包被抗原优化间接ELISA反应条件,确定其最佳抗原包被浓度为1μg·mL-1、血清稀释度为1∶200、血清作用时间60min、酶标二抗工作浓度为1∶6 000、二抗反应时间30min、底物显色时间10min。通过对138份临床血清样品检测发现其与HI试验结果符合率达88.41%。本研究建立的间接ELISA检测方法敏感、特异、准确,适用于临床样品的检测与血清流行病学调查。
[Abstract]:Goat parainfluenza virus type 3 can cause respiratory diseases in goats, and mixed infection with mycoplasma and bacteria causes widespread morbidity and mortality. The aim of this study was to establish an indirect ELISA antibody detection method based on N protein. Primers were designed to amplify the target gene Nand cloned into plasmid pET32a (). The recombinant plasmid pET32a-Nwas constructed and transformed into E. coli BL21 to induce the expression and identification of recombinant N protein. The detection method of ELISA antibody was established by optimizing the conditions, and the clinical samples were detected and compared with HI test. The recombinant plasmid was successfully constructed in this experiment. SDS-PAGE and Western blot showed that the protein was correctly expressed and had good antigenicity and specificity. The purified protein was used as the coating antigen to optimize the indirect ELISA reaction conditions. The best antigen coating concentration was 1 渭 g 路mL -1, the dilution of serum was 1: 200, the time of action was 60 min, the working concentration of enzyme labeled second antibody was 1: 6 000, the reaction time of the second antibody was 30 min, and the color reaction time of substrate was 10 min. 138 clinical serum samples were detected and found to be with HI. The coincidence rate of the test results was 88.41. The indirect ELISA detection method established in this study was sensitive. Specific, accurate, suitable for the detection of clinical samples and seroepidemiological investigation.
【作者单位】：江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室;南京农业大学动物医学院;
【基金】：江苏省农业科技自主创新资金项目[CX(14)2090] “十三五”国家重点研发计划(2016YFD0500900)
【分类号】：S852.654

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