兔出血症病毒间接ELISA抗体检测方法的建立

摘要

To establish an indirect ELISA for detecting rabbit haemorrhagic disease virus (RHDV) antibody,three regions of RHDV capsid protein VP60,namely VP60-1 (aa2-aa208),VP60-2 (aa174-aa425),and VP60-3 (aa342-aa579),were cloned into pET-32a (+) vector separately for prokaryotic expression.The expression of these three recombinant proteins with molecular weights of approximately 42 ku,47.5 ku and 45.6 ku were confirmed with SDS-PAGE,western blot and ELISA analysis.While VP60-1 had the best immunogenicity among VP60-1,VP60-2 and VP60-3.Then,an indirect ELISA was developed with the recombinant protein VP60-1 as coming antigen.The result showed that this method possessed high sensitivity,specificity and repeatability for detection of antibody against RHDV.The results of rabbits serurn in different immune periods detected by the ELISA were significantly correlated with that detected by HI.In conclusion,the ELISA detection method established in this study could be used for antibody titer evaluation for RHDV vaccination.%为建立兔出血症病毒(RHDV)的血清学检测方法,本实验通过生物信息学分析,将RHDV衣壳蛋白VP60的3个区域VP60-1 (aa2-aa208)、VP60-2 (aa174-aa425)和VP60-3 (aa342-aa579)分别进行原核表达,获得了大小为42 ku、47.5 ku和45.6 ku的重组蛋白.经ELISA和western blot试验分析显示3种重组蛋白均能够被RHDV阳性血清识别,但VP60-1较VP60-2和VP60-3具有更好的反应原性,因此以表达的重组蛋白VP60-1作为包被抗原建立了RHDV间接ELISA抗体检测方法.结果显示,该方法具有较好的敏感性、特异性和重复性,对不同免疫期兔血清的检测结果与HI呈显著相关性.对临床样品的检测结果表明该ELISA方法比HI试验更敏感.本研究以VP60-1为包被抗原建立的ELISA方法可以用于RHDV疫苗的免疫评估.