Preparation Of Monoclonal Antibody Against Nucleocapsid Protein Of Equine Arteritis Virus,Epitopes Identification And Establishmentand Application Of A Solid-phase Competitive ELISA

Equine viral arteritis （EVA） is a disease caused by Equine arteritis virus （EAV）, a contagious andreproductive viral infection among equids.In our study, the EAV Bucyrus srtain was used as the major material. Through virus production,RNA extraction and RT-PCR technique, the N gene was obtained. Using plasmid vector pET32a, weproduced a recombinant plasmid pET-N and then it was transferred into Rosetta （DE3）. By optimizinginducing environment, eventually N protein existing in90%supernatant was obtained and was purifiedby Ni-charged His Bind Resin. The result of Western blot indicates that N protein possess goodimmunogenicity and can be used as immunogen to produce monoclonal antibody （MAb）.BALB/C mice of6week-old were immunized with purified N protein and the spleen cells from theimmunized mice were fused with mouse myeloma cells SP2/0. By three cloning propagation, fivehybridoma cell lines stably secreting MAbs against N protein not His tag by western blot and IFA, werenamed1C11,2B1,2B3,3F11and8C4. The determination of competitive activity showed that only2B3and8C4share good competitive activity and can be used as the candidate MAb. To get high titerantibody ascites, these two kinds of hybridoma cells were magnified and injected BALB/C mice. Finally,8C4can be more competent to set up competitive ELISA（c-ELISA）.By using pepscan technique, five MAbs were used as probes to detect epitopes of N protein.Finally, three relatively epitopes were identified. Epitope¹⁸RRQPTSYND²⁶can react with2B3(¹⁸R→W¹⁸ï¼›¹⁹R→X¹⁹ï¼›²¹P→L²¹), epitope³⁸KPPAQP⁴³can co-react with2B1and3F11, epitope¹⁰¹QRKVAP¹⁰⁶can react well with1C11(¹⁰⁴V→I¹⁰⁴).In competitive ELISA, ELISA plates were coated by purified N protein and MAb8C4was usedas competitive antibody. The optimum reaction conditions were carried out by way of square titrationtest.For lack of essential EAV positive sera, we have to dilute EAV positive sera, which conserved in ourlab, in the fold of1:1.5,1:2,1:2.5,1:3,1:3.5,1:4. In our experiment, VN test and foreign EVA box wereused to determine the EAV positive serum samples. The result indicates that only six sera can beperformed in c-ELISA. It was also quite difficult to determine the cut-off point for lack of different titerantibody. Considering all the difficulties, six sera were used to confirm the ultimate limit. When theinhibition ratio locates between30%and40%, the result was considered doubtful; when inhibition ratiowas more than40%, the result was defined positive; when inhibition ratio was less than30%, the resultwas judged negative. The results of intra-repeatability and inter-repeatability showed that the CV can becontrolled in15%, which indicated that a good repeatability is endowed to this method. However,compared with VN test, we got a low sensitivity，for lack of enough positive samples. According to thenew method,100sera samples, which had been tested by VN and foreign box, were tested. The resultdemonstrated that three methods share complete coincidences. At the same time,300sera samplescollected randomly from Beijing, Guangzhou, Hebei, Qinghai, Xinjiang, Shandong, each provinceoffering50sera samples, was detected by c-ELISA and foreign box. The two diagnostic methods share the same negative result. Since equids belong to macrofauna, the sera component, in natuive, was verycomplicated and easy to be judged false positive. The c-ELISA compared with indirect ELISA canmake use of the specificity of MAb and prohibit false positive rate efficiently. Consequently, theinstallment of c-ELISA will set a firm foundation for the serum epidemiology investigation in China.EAV only has one serotype. Stallions once infected with EAV, will take along this virus all overthe life; however pregnant horses will suffer abortion, which cause substantial economy loss in horseconcerned industry every year. Along with the frequent horse trade communication and the rapiddevelopment of horse race industry, more and more importance has been attached to the work of how toprevent and diagnose EVA. Especially a rapid and precise serum diagnosis has become an urgent task todeal with.