基于RdRp蛋白的兔出血症病毒感染与免疫鉴别诊断ELISA方法的建立

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关键词：兔出血症病毒；RNA依赖性RNA聚合酶（ RdRp ）；间接ELISA中图分类号：S85.6 文献标志码：A 文章编号：0366-6964（026）01-0414-09

Abstract： To establish a diagnostic ELISA method for distinguishing RHDV infection and vaccination， the RdRp proteins of RHDV1 and RHDV2 were expressed by prokaryotic expression system.And these two soluble RdRp proteins were successully purified. RdRp protein was used as coated antigen，an indirect ELISA method was established to detect serum antibody against RdRp. After purification and restriction enzyme digestion，the RdRp protein without tag was obtained with a size of .Western blot analysis confirmed that both RdRp proteins reacted specifically with RHDV1 or RHDV2 infection sera. And there was no difference between the types of RdRp proteins. So RHDV2-RdRp was selected as a coating antigen at a coating concentration of 2.5μg⋅mL-1 .The optimal serum dilution was 1：100 and the incubation was 60min at 37∘C . The optimal dilution of enzyme-labeled antibody was 1：10 000 . The cut-off value was O.225. It was proved that the ELISA method has good specificity and sensitivity. The coeficient of variation in both intra-batch and interbatch repeatability test was less than 10% ，which indicating a good stability. The samples after RHDV challenge and the serum samples after genetic engineering vaccine immunization were tested， and the results showed that RHDV infection can induce the production of serum antibodies against RdRp protein， but the serum tests after genetic engineering vaccine immunization were negative. In this study，the ELISA method based on RHDV-RdRp protein can distinguish between genetic engineering vaccine immunity and RHDV infection，which provides an efective detection method for clinical screening of RHDV infection.

Keywords：RHDV;RNA-dependent RNA polymerase（RdRp）; indirect ELISA ∗ Corresponding authors： WANG Fang，E-mail： rwangfang@126. com； SONG Yanhua，E-mail ： songyanhua8507@126.com

兔出血症（rabbithaemorrhagicdisease，RHD）是一种兔的急性、烈性、高度致死性传染病，由兔出血症病毒（rabbithaemorrhagicdiseasevirus，RHDV）感染引起[1-2]，以呼吸系统出血，急性肝坏死，各实质器官水肿、淤血、出血为主要特征，其发病率及致死率较高[3]。（剩余14152字）