【作者】 张华；

【导师】 彭大新； 陈义平； 陈素娟；

【作者基本信息】 扬州大学 ， 预防兽医学， 2014， 硕士

【摘要】 传染性法氏囊病(Infectious bursal disease)是由传染性法氏囊病毒(Infectious bursal disease virus)引发的一种高度接触性传染病,感染对象主要为3-6周龄的幼鸡,法氏囊肿大或萎缩并伴随出血以及腿肌和胸肌出血为该病引起的主要病理变化,此外该疾病还可引起免疫抑制从而导致疫苗免疫失败以及其他疫病的发生,给全球养鸡业造成了重大的经济损失。检测传染性法氏囊病的方法很多,其中常用的血清学检测方法主要有琼脂扩散试验(AGP)和酶联免疫吸附试验(ELISA)。ELISA方法具有特异性强、敏感性高并且可实现高通量检测等优点,因此具有广阔的应用前景。目前市场上用于检测IBDV血清抗体的商品化ELISA试剂盒都是以全病毒作为包被抗原,本研究利用大肠杆菌原核表达纯化制备了IBDV VP2重组抗原,并以IBDV VP2上半段重组抗原作为包被抗原建立了用于检测鸡IBDV血清抗体的间接ELISA方法。一IBDV VP2基因原核表达参照Genbank中的IBDV VP2基因序列设计两对引物,通过RT-PCR技术扩增出VP2基因全长VP2(a11)以及上半段部分序列VP2(up),大小分别约为1350bp和750bp,纯化回收目的基因片段,连接克隆载体pMD18-T,构建重组克隆载体pMD18-T-VP2(all)和pMD18-T-VP2(up),经EcoR Ⅰ和Xho Ⅰ双酶切和基因测序鉴定。对重组克隆载体进行EcoR Ⅰ、Xho Ⅰ双酶切并纯化回收目的片段,通过连接和转化构建重组表达载体pET28a-VP2(all)和pET28a-VP2(up),经小提质粒和EcoR Ⅰ、Xho Ⅰ双酶切鉴定为阳性后,将阳性质粒转入表达菌株BL21中,VP2上半段基因用终浓度为1mM的IPTG,37℃诱导5h,VP2全长基因用终浓度为0.01g/mL的乳糖,37℃诱导5h,两种重组蛋白均获得表达,且均以包涵体形式存在,其中VP2全长蛋白大小约45kDa,表达量约占菌体蛋白的11%,VP2上半段蛋白大小约为27kDa,表达量约占菌体蛋白的30.1%。经过镍柱亲和层析后,获得纯化的重组VP2全长蛋白和VP2上半段蛋白,浓度分别为1.7mg/mL和2.5mg/mL。Western blot试验证明这两种重组蛋白均具有较好的免疫反应性,能与IBDV阳性血清发生特异性反应。二检测鸡血清中IBDV抗体的间接ELISA方法的建立通过方阵试验确定纯化的重组VP2上半段蛋白作为检测用抗原,每孔包被100ng,待检血清的最佳稀释度为1：100。通过对各种条件的优化,待检血清和酶标二抗的孵育时间均定为30min,酶标二抗的工作浓度选择为1：2500稀释。最后的判定标准为：当待检血清OD450≤0.6×ODP+0.4×ODN(ODp代表阳性对照血清OD450平均值,ODN代表阴性对照血清OD450平均值)判定为阴性,反之则判为阳性。对该方法的特异性检测试验中,H5、H7、H9亚型禽流感病毒阳性血清以及新城疫病毒阳性血清均被检测为阴性,说明该方法特异性良好。利用所建立的间接ELISA检测方法(VP2-ELISA)和IDEXX公司的IBDV血清抗体ELISA检测试剂盒平行检测156份临床鸡血清样品。以IDEXX公司的ELISA试剂盒检测结果作为参照,本实验所建立的间接ELISA检测方法的敏感性、特异性和符合率分别为95.65%(110/115)、90.24%(37/41)和94.23%(147/156),表明所建立的间接ELISA方法具有良好的特异性和敏感性,可用于鸡血清中IBDV抗体的检测。更多还原

【Abstract】 Infectious bursal disease (IBD) caused by infectious bursal disease virus, is a highly contagious viral disease of young chicken, which causes considerable economic losses to the poultry industry. IBD is characterized by muscle bleeding and bursal swelling or atrophy accompanied by hemorrhage, and3-6week old chickens are the susceptible targets. It is able to induce immune suppression, which leads to vaccination failure and occurrence of other disease. Agar gel precipitation (AGP) test and enzyme-linked immunosorbent assay (ELISA) are the main methods for diagnosing IBD in China. As advantages of specificity, sensitivity and high-throughput, the usage of ELISA is more and more widespread. But currently, there is still lack of a practical native ELISA kit for the detection of IBD. In this study, based on the first half of the VP2gene, an indirect ELISA method was established, which was able to detect serum antibodies against IBDV.1. The expressing of recombinant protein IBDV VP2According to the sequence of IBDV VP2in Genbank, two pairs of primers were designed targeting VP2(all) and VP2(up). The target fragments of1350bp and750bp were amplified by RT-PCR and cloned into pMD18-T vector respectively. Then, they were subcloned into the downstream of T7promoter of pET-28a (+) vector. The recombinant plasmids were transformed into E.coli BL21. Prior to the production of large-scale recombinant protein, a small scale optimization was carried out to estimate the optimum conditions for expression. An incubation temperature of37℃for6h with1mM of IPTG was the optimal condition for expression of VP2(up) protein. It was estimated that the VP2(up) protein constituted about30.1%of the total expressed protein. And the results of SDS-PAGE indicated that the size of VP2(up) was27kDa and the most of the protein was expressed in insoluble form. The VP2(all) was also successfully expressed in insoluble form following with lactose (10μg/mL) in E.coli BL21. It was about45kDa in size and constituted about11%of the total expressed protein.The concentrations of purified VP2(all) and VP2(up) protein were1.7mg/mL and2.5mg/mL respectively. The results of western blotting showed that both the recombinant proteins could react well with the positive serum against IBDV2. The establishment of an indirect ELISA for the detection of antibodies against IBDVThe purified VP2(up) protein (1μg/mL) was selected as the coating antigen and the optimal dilution of serum was1:100, which were determined by checkerboard titration. The incubation time of the serum and the HRP-conjugated antibody were both set to30min, and the optimal dilution of HRP-conjugated antibody was selected as1:2500. If OD450of the tested serum is less than or equal to0.6xODP+0.4×ODN (ODP is the mean of positive control serum, ODN is the mean of negative control serum), it is determined as negative. Otherwise, it is judged as positive. The test results of H5, H7, H9subtypes of avian influenza virus and Newcastle disease virus antiserum through this method show these four serums all are negative. So this method has a good specificity.A total of156clinical serum samples were detected parallel by VP2-ELISA and IDEXX ELISA kit. Compared to IDEXX ELISA kit, the coincidence rate, sensitivity and specificity of the VP2-ELISA were94.23%(147/156),95.65%(110/115) and90.24%(37/41) respectively. In summary, the indirect ELISA based on the recombinant protein VP2(up) is highly sensitive and specific. Therefore, this assay can be used to detect the antibody of chicken serum against IBDV and to evaluate the quality of IBDV vaccine. Otherwise the results of it are good for the formulation of immunization program.更多还原