鸭坦布苏病毒JM株E蛋白的截断表达及间接ELISA方法的建立
发布时间：2018-10-24 21:18
【摘要】：本研究利用RT-PCR扩增鸭坦布苏病毒(Duck Tembusu virus,DTMUV)JM株E基因截断片段(822 bp),并将其克隆至原核表达载体p ET32a(+),成功构建了重组质粒p ET32a-E。重组质粒转化大肠杆菌Rosseta,经IPTG诱导得到了高效表达。Western blot分析表明,重组蛋白能与DTMUV阳性血清发生特异性反应。将纯化好的DTMUV E蛋白作为包被抗原,建立了检测DTMUV血清抗体的间接ELISA方法。经过条件优化,确定了抗原最适包被浓度为0.093μg/孔,血清的最佳稀释度为1∶100。批内和批间重复试验的最大变异系数分别为3.53%和9.73%。用建立的ELISA方法对免疫鸭坦布苏病毒灭活疫苗的鸭血清和对照鸭血清进行抗体检测,同时与攻毒保护试验进行比较,两者的阳性符合率为86.67%,阴性符合率为100%。
[Abstract]:In this study, the truncated fragment of E gene (822 bp),) of (Duck Tembusu virus,DTMUV JM strain was amplified by RT-PCR and cloned into prokaryotic expression vector p ET32a (),. The recombinant plasmid p ET32a-E. was successfully constructed. The recombinant plasmid transformed into Escherichia coli Rosseta, was induced by IPTG to obtain the highly expressed. Western blot. The results showed that the recombinant protein could react specifically with the DTMUV positive serum. The purified DTMUV E protein was used as the coated antigen, and an indirect ELISA method for detection of DTMUV serum antibody was established. The optimum coating concentration of antigen was 0.093 渭 g / pore and the optimal dilution of serum was 1: 100. The maximum coefficients of variation were 3.53% and 9.73%, respectively. The ELISA method was used to detect the antibody of duck serum and control duck serum of the inactivated vaccine. The positive coincidence rate was 86.67 and the negative coincidence rate was 100.
【作者单位】： 广东省农业科学院动物卫生研究所
【基金】：广东省科技计划项目(2013B020307001、2014A040401049、2014A020208052、2015B020203003、2015B070701015、2016A020210049) 广州市科技计划项目(201510010250) 广东省农业科学院院长基金项目(201436、201412)
【分类号】：S852.65
[Abstract]:In this study, the truncated fragment of E gene (822 bp),) of (Duck Tembusu virus,DTMUV JM strain was amplified by RT-PCR and cloned into prokaryotic expression vector p ET32a (),. The recombinant plasmid p ET32a-E. was successfully constructed. The recombinant plasmid transformed into Escherichia coli Rosseta, was induced by IPTG to obtain the highly expressed. Western blot. The results showed that the recombinant protein could react specifically with the DTMUV positive serum. The purified DTMUV E protein was used as the coated antigen, and an indirect ELISA method for detection of DTMUV serum antibody was established. The optimum coating concentration of antigen was 0.093 渭 g / pore and the optimal dilution of serum was 1: 100. The maximum coefficients of variation were 3.53% and 9.73%, respectively. The ELISA method was used to detect the antibody of duck serum and control duck serum of the inactivated vaccine. The positive coincidence rate was 86.67 and the negative coincidence rate was 100.
【作者单位】： 广东省农业科学院动物卫生研究所
【基金】：广东省科技计划项目(2013B020307001、2014A040401049、2014A020208052、2015B020203003、2015B070701015、2016A020210049) 广州市科技计划项目(201510010250) 广东省农业科学院院长基金项目(201436、201412)
【分类号】：S852.65
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