The Prokaryotic Expression Of The Channel Catfish Virus Orf6 And Orf10 Gene And Their Function Analysis

Channel catfish virus (CCV) is the first herpesvirus which was discovered in fish. It was separated by Fijan from farmed channel catfish affected by channel catfish virus, and channel catfish is the natural host. CCV is a double-strand DNA virus which owns envelope, cause acute hemorrhagic toxemia and led channel catfish farming to a huge economic loss. The open reading frame ORF6 and ORF10 of channel catfish virus was thought to be envelope protein, which can participate in adsorption, invasion and transmission of virus, so the two genes were chosen to be analyzed and the main results were as following.1. CCO was recovered firstly and incubated in DMEM involved 10% FBS at 28℃.CCV was reproduced through infecting CCO, and then the genome of CCV was extracted.2. Specific primers were designed according to gene ORF6 and ORF10, the full length of ORF6 and ORF10 sequences were amplified by PCR which was 417 bp and 456 bp respectively. ORF6 and ORF10 were cloned to expressed vector pET-32a and the recombinant plasmid pET-32a-ORF6 and pET-32a-ORF10 was constructed successfully. The expressed proteins with theoretical molecular weight of 35.4 KD and 36.4 KD respectively were analysed by SDS-PAGE and were in the inclusion.3. The purified expressed proteins were used to immune rabbits separately and polyclonal antibodies were obtained. The results of Western Blot indicated that the polyclonal antibody of ORF10 could react specifically and was proved to be a structural protein of CCV, but the polyclonal antibody of ORF6 had no reaction. The results of neutralization experiment showed that ORF6 and ORF10 polyclonal antibodies can restrain the infection of CCV. It could be found the ability of protection of the two polyclonal antibodies became more and more weak with time lengthening, and the polyclonal antibody of ORF10 did better than the polyclonal antibody of ORF6.