Production, Characterization And Epitope Mapping Of Monoclonal Antibodies Against Different Subtypes Of Rabbit Haemorrhagic Disease Virus

Rabbit haemorrhagic disease virus(RHDV), is a calicivirus of the genus lagovirus that causes anacute, fulminating infectious and highly contagious disease in adult rabbits, namely rabbit haemorrhagicdisease(RHD). In 2010, a newly RHDV variant, designated RHDV2, was reported in France both inrabbits and in wild populations. Studies have shown that RHDV2 differs from RHDV1(traditionalRHDV) in terms of antigenic profile and genetic characteristics. Based on phylogenetic analyses of thevp60 gene, RHDV2 forms a separate branch, may indicate that RHDV2 is a new number of thelagovirus genus. At present, RHDV2 has not yet reported in China. Therefore, it becomes very urgentfor us to strength studies on the etiology, epidemiology, diagnosis and contol of the RHDV.VP60 is the major structural protein of RHDV, studies have demonstrated that VP60 protein is theimmune antigen of RHDV, playing an important role in the induction of immune response againstRHDV infection. In this study, vp60 gene of RHDV1 TP strain(Gen Bank: AF453761.1)was amplifiedand cloned into the p ET-32 a vector. RHDV2 10-32 strain’s vp60 gene(Gen Bank: HE800532.1)wassynthesized and also cloned into the p ET-32 a vector. Positive plasmids of p ET-R1-VP60 andp ET-R2-VP60 were transformed into E. coli BL21 Star(DE3)p Lys S cells, recombinant VP60 proteinp VP60-1 and p VP60-2 were successfully expressed when induced with IPTG. The fusion proteins werepurified by cutting the gel slices after staining with KCl. After antigenicity test by Western blot,p VP60-1 and p VP60-2 were then mixed with adjuvant separately and used as an immunogen. Spleencells of immunized BALB/c mice were collected and fused with SP2/0 myeloma cells. Based on aminoacids sequence comparison, six short peptides of higher difference were synthesized. p VP60-1,p VP60-2 and six synthesized peptides as well as RHDV1 TP strain were used as the detection antigen inindirect ELISA. At last, 20 strains of hybridoma cells against RHDV1 were filtered out after three timesof subcloning of which three can only recognize RHDV1, namely 1D6、1H2 and 3F2; 15 strains ofhybridoma cells against RHDV2 were filtered out of which four can only recognize RHDV2, namely1G2, 2C1, 3B7 and 5D6.VP60 was expressed by the eukaryotic expression system and was used to verify the recognitiondifference of 7 monoclonal antibodies(MAb) by indirect immunofluorescence assay(IFA) and Westernblot. The inducing ascites in vivo method was employed to produce MAbs in large amount.s VP60-1 and s VP60-2 were expressed using the Bac-to-Bac Baculovirus Expression System afterrecombinant RHDV1 vp60 baculovirus and recombinant RHDV2 vp60 baculovirus seeded sf9 cellseparately. Expressed s VP60-1 and s VP60-2 were used for IFA analysis with 7 MAbs. The vp60 ofRHDV1 and RHDV2 were cloned into eukaryotic expression vector pc DN3.1(+) and positive plasmidsof pc DNA-R1-VP60 and pc DNA-R1-VP60 were used to transfect Hela cell separately. 7 MAbs wereused as primary antibodies for Western blot and IFA analysis with e VP60-1 and e VP60-2 expressed inHela cell. Both IFA and Western blot showed that all 7 MAbs have recognition difference betweenRHDV1 and RHDV2. The epitope of 7MAbs was then defined by Western blot. Western blot showedthat RHDV1 VP60 recognition differences MAb 1D6 recognized the linear antigenic determinants of256 RWNGQ260, MAb 1H2 and 3F2 recognized the same sequence of 312VLQFW316. The minimalrecognition sequence for RHDV2 VP60 recognition differences MAb 2C1 appeared to be 324ADNPIS329,MAb 1G2 、 3B7 and 5D6 to be the same sequence of 294AIDHD298(different amino acids betweenRHDV1 VP60 and RHDV2 VP60 protein are identified by italic). An analysis to assess the conservationof identified recognition differences MAb epitopes among members of the genus Lagovirus wasperformed. The result showed that epitopes of RHDV1 VP60 recognition differences MAb are highlyconserved among RHDV1(98%), whereas epitopes of RHDV2 VP60 recognition differences MAb arecompletely conserved among RHDV2. These results indicate that the screened MAb’s recognitiondifferences ability is quite stable and able to distinguish different subtypes of RHDV, are type-specificMAbs.This research prepared RHDV type-specific monoclonal antibodies by hybridoma fusion andprecisely mapped epitopes of type-specific monoclonal antibody. Type-specific monoclonal antibodypreparation laid the foundation for the establishment of RHDV subtype-specific detection methods, andhas important significance for RHDV epidemiological investigation and phylogenetic analysis.