【作者】 刘立峰；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 1997年以来，在我国许多地区爆发了由鹅副粘病毒(Goose Paramyxovirus，GPMV)引起的鹅的烈性传染病。该病是以消化道和呼吸道病变为主要特征，具有高度发病率和死亡率，给养鹅业带来了很大的损失。国内外学者先后建立了琼脂扩散试验、中和试验、血凝抑制试验等血清学诊断方法，在一定程度上为防治鹅副粘病毒病发挥了重要作用，但同时也存在灵敏度差、操作烦琐、检测所需时间长等不足，已不适应更有效地控制该病的需要。 本论文将鹅副粘病毒NP基因的重组质粒pGEX-6P-NP转化到工程菌BL21(DE3)pLysS内，重组NP蛋白在大肠杆菌中获得了表达，并且进行了切胶纯化。用表达的融合蛋白作为包被抗原，以灭活油苗免疫制得的鹅血清做为一抗，HRP标记的兔抗鹅血清作为二抗，并确定了ELISA最佳工作条件，即抗原包被浓度为54μg／ml，4℃包被过夜，血清(1：800)和酶标兔抗鹅(1：1500)分别在37℃1h，底物溶液37℃显色20min。对各项条件和参数进行了优化，为开发标准化诊断试剂盒奠定了坚实的基础。通过对检测抗原的交叉试验及阻断试验均证明，检测抗原只与抗GPMV抗体发生特异性结合。健康非免疫鹅血清及SPF鸡血清的检测均出现阴性结果。鸡胚尿囊液中的GPMV可阻断检测抗原与相应阳性血清的结合，证明该检测抗原的特异性是可靠的。所建立的方法重复性好、特异性强、灵敏度高。可用于GPMV流行病学的调查和GPMV灭活苗免疫后血清抗体水平的检测。更多还原

【Abstract】 A new viral disease whose pathogen was goose paramyxovirus has outbroken in many areas of china since 1997. Characteristic of the disease is the lesion mainly in the digestive, respiratory tract and high mortality, causes serious loss in goose breeding industry. The routine diagnostic methods for GPMV were the Agargel precipitin assays (AP), Serum-neutralization test (NT) , have been established. These methods have played important role in control of GPMV, but which has lower sensitivity and take longer time. These methods have not been suitable for the needs of efficient control of GPMV.The study is that the recombinant expression plasmid pGEX-6P-NP was transformed into BL21(DE3)pLysS. The recombinant NP protein was expressed in E.coli and purified. An indirect ELISA was developed with the purified protein as coating antigen, goose serum which was made by inactivated vaccine as antibody and HRP conjugated rabbit anti-goose IgG as anti-antibody. The optimal conditions were that antigen is coated at 4℃ overnight and its concentration is 54μg/ml, serm sample (1:800) and HRP conjugated rabbit anti-goose IgG(1:1500) are incuabtaed at 37℃ for 60 min, the substrate of ELISA is incuabated at 37℃ for 20min. Every condition and parameter is optimized, then settle a good foundation for standardizing diagnostic kit. It is testified that the coating antigen only react with GPMV antibody by crossing assay and blockage assay. The negative result is shown with serums from healthy goose and SPF chicken. The reaction between antigen and positive serum can blocked by GPMV in chick embryo which proves specificity of the antigen is faithful. The developed method has good specificity, sensitivity and repeatability that can be used as epidemiologic survey and detection of antibody after being immunized by GPMV inactivated vaccine.更多还原