【作者】 郝春丽；

【导师】 王泽霖； 王新卫；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 IBV血清型众多,不同血清型间交叉保护差,给诊断与防制带来很大的困难;为建立一种更为简便、快速、群特异性、能用于大面积检测抗体的方法,来补充中和试验和HI试验,以评价疫苗的免疫效果,本研究将对NP-ELISA和S1-ELISA进行探讨。本研究利用RT-PCR技术成功地扩增了传染性支气管炎病毒M41株的NP基因,克隆并重组于原核表达载体pET-28a,经PCR、酶切鉴定及序列测定成功地构建了重组表达载体pET-NP。重组菌pET-NP经IPTG诱导,获得了高效表达,分子量约为49kDa,且以可溶性蛋白形式存在。重组菌pET-NP经大量诱导表达后,收集菌体,超声波裂解,取上清透析后,采用Ni亲和层析柱纯化重组NP蛋白,经12%的SDS-PAGE分析,表明获得了纯度较高的重组NP蛋白。用纯化的NP蛋白作包被抗原,建立了检测IBV抗体的NP-ELISA。抗原最佳包被浓度为1.45μg/孔,待检血清最佳稀释度为1:80,酶标二抗最佳工作浓度为1:800。确定阳性判定标准为0D450≥0.270。NP-ELISA与AIV H9、H5、H7、IBD、ND、EDS₇₆的标准阳性血清不发生交叉反应,具有良好的特异性,且灵敏度高、重复性好,但试验表明与HI试验、攻毒保护试验结果没有相关性,故不能用于IB免疫效果的评价方法。本研究利用RT-PCR技术成功地获得了完整的S1基因（S1-1）,片段大小为1614bp。在分析全长S1基因的基础上,又扩增了去掉信号肽及包含主要抗原位点的S1-2和S1-3片段,大小分别为1533bp和801bp。将三个片段分别克隆并重组于原核表达载体pGEX-6P-1,构建了pGEX-S1-1、pGEX-S1-2、pGEX-S1-3重组原核表达载体,经PCR、酶切鉴定及序列测定证明三个基因片段均正确地插入到表达载体中,且阅读框正确。三种重组质粒分别转化于宿主菌BL21（DE3）,经IPTG进行诱导表达,表达产物经12%的SDS-PAGE电泳分析,结果表明,重组质粒pGEX-S1-1和pGEX-S1-3没有得到有效表达,而重组质粒pGEX-S1-2获得了表达,表达产物以包涵体形式存在,表达产物的分子量约为80kDa,与理论上推测的分子量大小一致。表达产物经Western-blotting分析表明,该表达蛋白能与M41株标准阳性血清反应,说明此表达蛋白具有良好的生物学活性。S1蛋白是最重要的保护性抗原,可诱导机体产生中和、血凝抑制及保护性抗体,通过实现S1蛋白的有效表达,并建立S1-ELISA,探讨S1-ELISA效价与攻毒保护间的关系,有望替代HI试验和中和试验,用于免疫后抗体的检测和疫苗质量的评价,S1蛋白的表达也可为S1蛋白结构与功能的深入研究和基因工程疫苗的研制奠定良好基础。更多还原

【Abstract】 IBV has many different serotypes and clinical situations. Weak cross protection appear between different serotypes. Resently, IBV seriously hinder the development of poulty industry with the occurrence of new variant strains and serotypes. In order to affords strong specificity, high sensitivity ,excellent coincidence and can apply to detect the field sera samples in bulk and procvide an effective method for diagnosis and immunal monitoring of IB ,and also can incomplement VN test and HI test to evaluate the effect of the vaccine. we will do the research on NP-ELISA and S1-ELISA.The NP gene was obtained by RT-PCR , which nucleotide sequence is 1614bp in length .Then NP gene was cloned and recombined to the pET28a to contrast the expressed vector pET-NP. pET-NP was proved to be right and was induced by IPTG in bulk and was clearaged by ultrasonic wave to obtained the solubily N protein, The expessed N protein was purified by HisTrap HP Kit. The result demonsrated that the purified NP was suitable to be ELISA antigen. The purified N protein was used to establish an indirect ELISA assay for the detection of antibody againt IBV. The optimal concentration of coating antigen was1.45μg/mL, and the optimal dilution of serum and HRP labeled rabbit anti-chicken was 1:80 and 1:800. The border of negative and positive serum in OD₄₅₀ value was 0.270,when the OD₄₅₀ value was equal or over 0.27,it was positive, or else negative. NP-ELISA had no reaction to the positive serums of AIV H9、H5、H7、ND、IBD and EDS₇₆ ,which directed that the expressed N protein was only reacted to the serum of IBV. The result have no relativity bewent NP-ELISA and HI test. The results above indicated the NP-ELISA not only affords strong specificity, high sensitivity .excellent coincidence and can apply to detect the field sera samples in bulk.Complete S1 gene of Avian infectious bronchitis Virus M41 strain was amplified by RT-PCR, which nucleotide sequence is 1614bp in length. According to analysis of S1 pprotein, two pairs of primers were designed to obtain the 1533bp and 801bp of S1 gene, which one was deletaded the signal peptide and another included important antibody sites. The four fragments were cloned into the pGEM-T easy vector to construct the recombinant clone plasmids. The plasmids were proved to be true by PCR, restriction endonuclease analysis and the analysis of nucleotide sequences. Then the four fragments were recombinated into prokaryotic expression vectors pGEX-6P-1 to construct the expression vectors pGEX-S1-2、pGEX-S1-3, which were identified to be true by PCR, restriction endonuclease analysis and the analysis of nucleotide sequences, and then transformated into E.coli BL21 （DE3）. The recombinant expression plasmid was induced by 1mmol/L IPTG at 37℃for 5 hours and analyzed by 12% SDS-PAGE. The results described that the recombined vector （pGEX-S1-2） was lowerly expressed in inclusion body which molecular weight was about 80 KDAa as expected by the analysis of SDS-PAGE. The result of western-blot demensrated the fusion proteins of S1 have partical antigen, which indicats the expression products have clinical value as the diagnosis antigen and gene engineering vaccine.更多还原