双抗体夹心ELISA检测传染性支气管炎病毒方法的建立

发布时间：2018-04-12 19:56

本文选题：传染性支气管炎 + N蛋白　；参考：《扬州大学》2017年硕士论文

【摘要】：传染性支气管炎病毒(infectious bronchitis virus,IBV)可引起鸡的一种急性、高度接触传染性的呼吸道和泌尿生殖道疾病——鸡传染性支气管炎(infectious bronchitis,IB)。自1931年被首次报道后,IB在世界各地均有发生,IBV现已衍生出多种血清型。IBV可与其它病原体混合感染,引起严重的并发症,给养禽业带来巨大损失。IBV抗原性复杂,易发生基因重组和点突变,而且不同血清型间的交叉保护效力低,使IB的防控极具挑战性,因此,开发简便、快速的诊断方法,对IB的防控非常重要。核衣壳蛋白(N蛋白)是IBV的主要结构蛋白,相对较保守,本研究针对N蛋白制备了兔多克隆抗体(PcAb)和鼠单克隆抗体(MAb),建立了检测IBV的双抗体夹心ELISA检测方法,为IBV的诊断提供了一种行之有效的手段。1.针对IBVN蛋白的兔多克隆抗体和鼠单克隆抗体的制备本研究以IBVQXL毒株为基础,对其N蛋白的序列进行抗原性分析,用PCR方法扩增抗原性较强区域对应的N基因片段,然后将产物连至克隆载体上,经基因测序鉴定正确后,再以pET-32a(+)为载体,构建表达N蛋白片段的重组原核表达质粒,转化宿主菌BL21(DE3)后诱导表达并纯化,获得的N蛋白片段用作免疫原,分别免疫成年兔和6-8周龄的BALB/C小鼠。当免疫兔的血清效价达到1:104时,心脏采血制备抗N蛋白的PcAb血清;当免疫鼠的血清效价达到1:104时,取脾脏与SP2/0细胞融合,利用间接ELISA鉴定、筛选杂交瘤细胞,最终获得四株能稳定分泌抗N蛋白MAb的细胞株6F4、7D8、3G2、5C9,制备腹水并鉴定、纯化后,进一步用Western blot鉴定,结果表明获得的单抗可与N蛋白特异结合。2.检测IBV的双抗体夹心ELISA方法的建立本研究以制备的PcAb为包被抗体、MAb为检测抗体,建立检测IBV的双抗体夹心ELISA方法。优化后的反应条件为:包被抗体最佳稀释度为1:64000,检测抗体最佳稀释度为1:4000,封闭液为1%BSA-PBST,酶标抗体稀释度为1:10000,显色底物作用时间为15 min。特异性实验表明,该方法对禽流感病毒(AIV)、新城疫病毒(NDV)及禽腺病毒(FAdV)无交叉反应。重复性实验表明,该方法的批内、批间变异系数均小于8%。该方法与检测IBV的PCR方法的阳性符合率为95.9%,阴性符合率为85.2%。总之,本研究制备的抗体可以和IBV的N蛋白特异性反应,以此为基础建立的双抗体夹心ELISA检测IBV方法的特异性好、可重复,且检测结果可靠,为IB防控提供了良好的诊断方法。
[Abstract]:Infectious bronchitis virus (IBV) can cause acute and highly contagious respiratory and urogenital diseases in chickens.Since it was first reported in 1931, IBV has been found in various parts of the world. IBV can be mixed with other pathogens, causing serious complications and causing huge losses to poultry industry. IBV antigenicity is complex.Gene recombination and point mutation are easy to occur, and the cross-protection efficacy between different serotypes is low, which makes the prevention and control of IB extremely challenging. Therefore, it is very important to develop a simple and rapid diagnostic method for the prevention and control of IB.Nucleocapsid protein (N) is the main structural protein of IBV, which is relatively conserved. In this study, rabbit polyclonal antibody (PcAb) and mouse monoclonal antibody (McAb) were prepared for N protein, and a double antibody sandwich ELISA method was established for the detection of IBV.It provides an effective method for the diagnosis of IBV.The preparation of rabbit polyclonal antibody and mouse monoclonal antibody against IBVN protein. Based on the IBVQXL strain, the sequence of N protein was analyzed antigenically, and the N gene fragment corresponding to the region with strong antigenicity was amplified by PCR.The recombinant prokaryotic expression plasmid expressing N protein fragment was constructed by using pET-32a () as vector. The recombinant plasmid was transformed into host strain BL21DE3) and then induced and purified.The obtained N protein fragments were used as immunogen to immunize adult rabbits and 6-8 week old BALB/C mice respectively.When the titer of serum of immunized rabbits reached 1: 104, the PcAb serum of anti-N protein was obtained from the heart, and when the titer of serum of immunized mice reached 1: 104, the spleen was fused with SP2/0 cells, and the hybridoma cells were screened by indirect ELISA identification.Finally, four cell lines that stably secreted anti-N protein MAb were obtained. Ascites were prepared and identified by Western blot. The results showed that the obtained McAbs could specifically bind to N protein.Establishment of a double antibody sandwich ELISA method for detection of IBV A double antibody sandwich ELISA method was established for the detection of IBV using the prepared PcAb as the coated antibody to detect the antibody.The optimized reaction conditions were as follows: the best dilution of coated antibody was 1: 64000, the best dilution of antibody was 1: 4000, the blocking solution was 1BSA-PBST, the dilution of enzyme-labeled antibody was 1: 10000and the reaction time of chromogenic substrate was 15 min.The specificity test showed that the method had no cross reaction against avian influenza virus (AIV), Newcastle disease virus (NDV) and avian adenovirus (FAdV).The repeatability experiment showed that the coefficient of variation was less than 8%.The positive coincidence rate and negative coincidence rate between this method and PCR method were 95.9 and 85.2, respectively.In a word, the antibody prepared in this study can react with the N protein of IBV, and the double antibody sandwich ELISA based on this method has good specificity, repeatability and reliable detection results, which provides a good diagnostic method for IB prevention and control.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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