Construction Of Adenovirus CELO Vector And Expression Of The Partial Fragment Of Fiber 1 Gene

Chicken embryo lethal orphan virus (CELO) is classified as a fowl adenovirus type 1 (FAV-1) and is the major subject of avian adenovirology for many years. And now it is being developed as a vector system for vaccine design. The fiber 1 and fiber 2 genes of CELO are responsible for viral virulence and immunogenicity. The CELO genome with disrupted fiber 1 gene produced virus that was capable of infect chicken cells, but lost the ability to efficiently transduce human cells. Based on this character, a replication adenovirus vector was generated. And the fiber 1 carboxy-terminal knob-s was expressed in E. coli system.A fragment 3 611bp (27 150bp to 30 760bp) (named Fc) comprising of fiber 1 gene and flanking sequences, was amplified by PCR from the genome of CELO. cloned into pMD18-T. yielding pT-Fc. Plasmid pT-Fc was digested with Xhol, resulting in a deletion of 592bp. The CMV-eGFP expression cassette was released from plasmid pEGFP-N1 using AflII and Asel. Then the two fragments were blunted and religated, obtainting pTF-CMV-eGFP. The pT-Fc-CMV-eGFP and CELO genomic DNA were used to cotransfect 293-T cells. Then the recombinant virus was rescued by infecting chicken embryoed fibroblasts (CEFs) or chicken kidney cells (CKCs), giving rise to a recombinant virus CELOF-eGFP, which was identified by green fluorescence and PCR.The 807bp immunodominant region of CELO fiber 1 protein (AF807) was amplified from Fc fragment and cloned into a prokaryotic expression vector pGEX-6p-l to obtain pGEX-AF807. The target fusion protein was produced by inducing pGEX-AF807-transformed E. coli cells with IPTG And SDS-PAGE analysis showed that the expressed fusion protein existed in form of inclusive bodies and its molecular weight was about 54.6ku. The fusion protein accounts for 35.7% of the total bacterial proteins. Western blot analysis showed that the protein has good antigenicity.The CELOV vector may be very useful both for in vitro and in vivo gene therapy applications. And the expressed protein can be used as antigen in CELO serodiagnosis and differential diagnosis accompanying the vector vaccine.