Isolation And Identification Of Chicken Embryo Lethal Orphan Virus (Celov) And Prokaryotic Expression Of Protein â…¢a And Analysis Of Antigenicity

Duck viral hepatitis seed virus(E52 strain) was reproduced by non- immunitied embryo. But it was contaminated by another virus which can infect chicken. The chicken was sub-healthy and not sick. However contaminated virus can disseminate embryo directly by hens. The virus was purified by PEG6000, Sephadex G100 gel chromatography and dialysis. Then it was observed by EM and measured up to the morphology of adenovirus.While observing duck viral hepatitis seed virus in chicken embryonic allantochoroin liquid, another symmetry nonenveloped icosahedral viron with a diameter of 70 to 80 nm was found. In order to investigating this contaminating virus, we select four random primers to amplify this unknown virus by RT-PCR method and PCR method respectively, that resulted three DNA fragments, showing 99.5%, 99.6% and 99.5% homologue respectively with the genome of adenovirus type I, or chicken embryo orphan lethal virus (CELOV) during the sequencing analysis ( one cycle reaction) for them. The result suggested that duck viral hepatitis seed virus was contaminated by chicken embryo lethal orphan virus (CELOV).Based on Ⅲa gene sequence data, a fragment 1 728 bp was amplified by PCR from the genome of CELOV and cloned into plasmid pGEM T Easy. The result of PCR showed that the designed fragment was amplified with expected molecular weight and was subcloned into a prokaryotic expression vector pET-32a(+). The recombinant plasmid was named as pET Ⅲa .Expression vector of protein Ⅲa was transformed into E.coli strain BL21,the host was named BL Ⅲa, After inducing by IPTG,the expression production experiment showed that fused protein TrxA-Ⅲa was expressed in BL21 Ⅲa with expected molecular weight 77KD.the result of Western blot showed that Trix- Ⅲ a had the antigentic characteristic of CELOV. Then the fused protein was used to immunize the mouse and positive antibody was obtained. The antigenicity of protein Ⅲa was tested by IFA. Further study is on the way.Incubation of Trix-Ⅲa for expression was studied. The optimized conditions for induction of Trix-Ⅲ a was: the concentration of the BL21 Ⅲa before induction amounted to OD6000.4-0.6.,37℃,0.2 mM IPTG, incubating 2-3hrs after incubation. The Trix-Ⅲa protein amounted to about 33% of the total protein and was expressed not as inclusion bodies.The purification process of the recombinant Trix-Ⅲa fusion protein was studied. The fusion protein was purified by Metal chelated chromatography. The production of purification was lyophilized as antigen for future study.