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-OmpA_1-2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-OmpA_1-2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-OmpA_1-2融合表达蛋白免疫鸭,采血分离血清。GST-OmpA_1-2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-OmpA_1-2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-OmpA_1-2表达蛋白对免疫鸭不能提供完全保护。
研究认为GST-OmpA_1-2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-OmpA_1-2可望作为包被抗原用于RA的诊断试剂盒的制备。
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-OmpA_1-2) 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-OmpA_1-2 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-OmpA_1-2 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-OmpA_1-2 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-OmpA_1-2 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-OmpA_1-2 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.
引文
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