The Study Of Characterization And Loction Of Envelope Protein Of The Channel Catfish Virus

Channel catfish virus (CCV) was the first herpesvirus which was discovered in fish. Because orf10was predicted to encode a type I membrane protein with a potential, cleavable signal sequence (amino acids, aa1-24) and a transmembrane domain (amino acids, aa101-123), as determined by the computer program SignaIP3.0(Bendtsen et al.2004) and SOSUI (Hirokawa et al.1998), it was expressed in E. coli and used as an antigen to prepare an antibody. This study was disparted parts:first member studied function characteristic and location; second member studied the method of DNA probe detection of the channel catfish virus and immunity of production. the main results were as following.1. In this study, we have successfully cloned the orf10gene of CCV. orf10is456bp and has no significant homology with other known viruses in the Alloherpesvirus family by sequence alignment. The estimated molecular mass of the protein by western blot analysis was approximately35kDa, which is much greater than the theoretical molecular mass (16kDa). This difference may be attributed to post-translational modifications or interactions with other envelope proteins. Also there were two bands that differed in size by about2kDa present in the intact virion and envelope lanes of the western blot, which might be due to disulfide bonding and/or glycosylation for ORF10was predicted to contain two potental N-glycosylted sites at Asn64and Asn84. As in prokaryotic expression system, the product of orf10gene displayed the equal molecular mass with expected size for lacking the posttranslational modification.2. In this study, we have successfully identified its location in virions.The product of the orf10gene was identified to be an envelope protein, based on the results of western blotting assay and immuno-electron microscopy.3. The results of the neutralization assay showed that CCV infection was reduced, due to the steric hindrance or the blocking of the binding with its cellar receptor by the primary antibody.These results suggested that orf10was also involved in the entry of CCV, although its exact mechanism was not clear. More interesting would be a later consideration of whether neutralizing this one epitope was likely to be sufficient to protect fish.4. ORF6envelope protein using new expression carrier had good immunity.5. A probe and a pair of primers were designed based on the orf6of CCV.5'end of primer was labeled by biotin,3'end of probe was labeled by digoxigenin, sample was captured by streptoavidin-biotin function. The hybridization with sample and probe was carried out on PCR. Then the hybridized product was detected with a AP-labeled anti-DIG antibody and PNPP substrate.6. Established PCR-ELISA measure method had characteristic of strong special, good repetition and high sensitivity. Variance coefficient of repetition measure to a patch was less than10%; Variance coefficient of repetition measure among patchs was less than15%. Sensitivity of this method was5fg CCV DNA.