【作者】 刘文华；

【导师】 苏敬良； 赵继勋； 刘金华；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 本试验利用PCR技术扩增了标准血清2型RA的42KDa外膜蛋白基因片段（OmpA-2）1045bp,与原核表达载体pGEX-4T-1连接构建重组表达载体,转入大肠杆菌BL21菌株中。在1mM的IPTG诱导下,表达融合蛋白,诱导后5h蛋白表达量达到高峰。融合表达蛋白GST-OmpA₁-2的分子量约为65KDa,以包涵体形式存在。 以脱氧胆酸钠（DOC）洗涤包涵体,然后用N—十二烷基肌氨酸钠（SKL）溶解,将溶解包涵体进行透析复性。分别用亲和层析法、电洗脱法和研磨法对GST-OmpA₁-2融合蛋白进行了纯化,亲和层析纯化效果不佳,电洗脱法和研磨法则获得了较好的纯化效果。 将GST-OmpA₁-2融合表达蛋白免疫鸭,采血分离血清。GST-OmpA₁-2以0.85μg/mL的浓度包被酶标反应板,鸭抗2型RA阳性血清进行200×稀释,HRP-山羊抗鸭IgG作5000×稀释,建立间接ELISA方法检测免疫鸭的抗体水平。间接ELISA和Western-blotting试验结果均表明GST-OmpA₁-2表达蛋白具有良好的反应原性。以2型RA强毒攻击免疫鸭,结果表明GST-OmpA₁-2表达蛋白对免疫鸭不能提供完全保护。 研究认为GST-OmpA₁-2表达蛋白具有良好的反应原性,阻断试验表明不存在血清型特异性,所以,GST-OmpA₁-2可望作为包被抗原用于RA的诊断试剂盒的制备。更多还原

【Abstract】 In this study, 1045bp gene fragment of the 42KDa OmpA of RA serotype 2 （OmpA-2） was amplified by PCR, then combined with pGEX-4T-l vector, and as a sequence, the recombinant plasmid containing OmpA-2 gene fragment was obtained. The recombinant fusion protein （GST-OmpA₁-2） was expressed at highest level in E.coli BL21 induced by 1mmol/L IPTG for 5h in the form of inclusion bodies. The molecular weights of GST-OmpA₁-2 is about 65 KDa.The inclusion bodies were washed with Deoxycholic acid Sodium Salt （DOC） for several times, and then dissolved in N-Lauroyl Sarcosine Sodium （SKL） for denaturant. After that, a good renature result was obtained by dialysis.The purification of GST-OmpA₁-2 was done by affinity-chromatography of Glutathione SepHarose 4B, electrodialysis method and abrasion method, while only the last two methods attained perfect results.The GST-OmpA₁-2 was used to immunize duck. The indirect ELISA was established to detect the level of antibody of vaccinated ducks with procedure as follows: reaction plate coating by GST-OmpA₁-2 of 0.85μg/mL, 200 X dilution of RA positive serum and then 5000 X diluted HRP-goat antiduck IgG The results of indirect ELISA and Western-blotting showed that GST-OmpAi-2 had good antigenicity.GST-OmpA₁-2 showed not absolute protection for the cluck when challenged with RA Type 2 to the immune ducks.From the study, it can be concluded that GST-OmpA]-2 had good antigenicity and showed no specificity between vary serotypes of RA by blocking ELISA, so application to the clinical detection of RA infection and serological investigate is possible.更多还原