Construction,Identification And Expression Of The Recombinant Adenovirus Vectors Encoding Porcine Circovirus Type 2 Functional Gene Cap/rep/rep’

Porcine circovirus type 2（PCV2）is the causative pathogen of porcine circovirus-associated diseases（PCVAD）,which is a multi-systemic dysfunctional disease characterized by swollen systemic lymphadenopathy in pigs.It causes significant economic losses in swine-producing areas and countries.Previous study has shown that suppressing the ORF1 and ORF2 of PCV2 can significantly inhibit the replication of PCV2.As known,ORF1 encodes the rep and rep’proteins which are associated with viral replication,while ORF2 encodes the structural protein cap which is also the major immunogenic protein of PCV2.Although a series of investigations on PCV2-related functional proteins have been conducted,the mechanisms underlying low PCV2 virus replication in mammalian cells largely remain undetermined,thereby limiting the development of vaccine against PCV2.Adenovirus gene delivery technology has become one of the most widely used live viral gene delivery vector due to its unique properties including high transduction efficiency,wide range of host cells and the ability to infect cells at different stages.To this end,in this thesis I adopt the adeasy1 system of adenovirus gene transfer technology,and select the popular strain PCV2b,and separately construct recombinant adenoviral vectors expressing cap/rep/rep’genes,and then transfect there vectors into 293T cells for virus packaging and expansion,finally perform the identification and titration for these recombinant adenoviruses,respectively.The results showed that three recombinant adenoviral vectors were successfully constructed.Fluorescence observation,PCR,IFA and Western blotting could detect the expression of the target genes in 293T cells,suggesting the recombinant adenoviruses were successfully packaged and the titers of them could be up to 2×10¹⁰GFU/m L.The construction of three recombinant adenovirus vectors,the packaging and titration of the corresponding viruses,the expression of the three functional proteins of PCV2were confirmed in the initial study.Next,the overexpression of PCV2 functional proteins in the host cells which are inoculated with individual or combined adenoviruses,will facilitate to reveal the important roles for the three functional proteins in the replication of PCV2,provide the foundation for enhancing PCV2replication,and pave the way for the development of more effective vaccines against PCV2.