Preparation Of Monoclonal Antibodies Against Protein VP1 Of Pancreatitis-type DHAV-1

The pathogen of this experiment is pancreatitis-type Duck Hepatitis A Virus Type 1(DHAV-1).The main lesions of it are pancreatic change to yellow and bleeding in Muscovy Duckling, they are different with the typical Duck Hepatitis A Virus Type 1(DHAV-1).Compared with DHAV-1 reference strains from GenBank, genome of MPZJ1206 isolate shared 94.1%-99.1% homology with DHAV-1. But the homology of deduced amino acid sequence of VP1 gene between MPZJ1206 isolate and DHAV-1 shows similarity score were 96.8%-97.5%.The experimental design based on its VP1 gene sequence specific primer pair, the VP1 gene fragment was amplified by RT-PCR, and the amplified fragment was recovered and purified connected to pMD18-T cloning vector, and get connected to the product transformed into competent cells of E.coli Trans5a.After correcting the sequence, pMD18-T-VP1 were digested by BamH I and Xho I double enzyme.The VP1 gene was subcloned into pGEX-6P-1, and transformed into E.coli BL21, the positive clones were screened by double enzyme digestion.The positive ones were induced by IPTG and optimized the condition.The expression protein were analyzed by SDS-PAGE.The best results was induced 5 hours at 37℃, the VP1 protein was expressed in the form of inclusion and its molecular weight was about 53 kDa, which is equal to that of the expected product.The fusion protein activity was identity by Western-blot.The protein used to immunize BALB/c mice, indirect ELISA screening method were established.Within 72h after the last immunization the SP2/0 which is in the logarithmic growth phase were fused with spleen cells of immunized mice. Indirect ELISA was used to screene hybridoma cells.Two positive hybridoma cells were screened, named as 1A2,5G3.We also identified the stability of McAb subclasses, ascites titer and specificity.From the results we can see that the monoclonal antibody has well stability, high titer good specificity.The antibody titer of 1A2 cell strains against DHAV-1 is 1: 25×103 and 5G3 is 1:211×103.Identification of subgroup showed that they both belonged to IgGl, κ light chain.The Western-blot shows that these McAbs were specific only to VP1 protein.The experimentally obtained DHAV-1 monoclonal antibody and lays a firm foudation for for the treatment and the development kit to detect it.