Research Of Recombinant Adenovirus Co-expressing The Capsid Protein Of Porcine Circovirus 2(PCV2) And Immune Enhancing Factor The C Terminal Of Yersinia Invasin Gene

Porcine circovirus diseases(PCVD), caused by porcine circovirus type II(PCV2), is a range of infectious diseases that hinders the booming pig industry seriously. The main measure for preventing porcine circovirus diseases is vaccination. The porcine circovirus type 2 vaccines extensive used are inactivated vaccines that have a better effect. However, there are some problems for it, including low virus drop degree, the need for virus concentration, and expensive. Developing a new PCV2 vaccine with higher titer, perfect immune effect and satisfactory safety is a voguish research direction. As possessing advantages of simple preparation, high virus drops, and durable immune stimulation, adenovirus-vectored live vaccines attracts much attention for study in PCV genetic engineering vaccine. The recombinant virus constructing strategy is inserting PCV2 capsid protein gene into virus genome. The C terminal of Yersinia invasin gene(InvC) is part of outer membrane protein that can improve immune effect through enhancing the host cells` ability to absorb antigens. For a new type of PCV2 adnovirus-vector vaccine, this study constructed a recombinant adenovirus co-expressing PCV2 Cap gene and InvC gene, which means displaying objective antigen and immunological enhancing factor at the same time. Then animals was immuned with it. And immune efficacy was evaluated. The following results obtained in this study:First, the recombinant adenovirus co-expressing PCV2 Cap gene and InvC gene was constructed. Having been embedded with PCV2 Cap gene and InvC gene successively, the recombinant shuttle plasmid was transformed into competent cell BJ5183 to make the framework plasmid of recombinant adnovirus. The gotted framework plasmid was transfected into HEK293 cells to make recombinant adnovirus named rAd-Cap-InvC. PCR, RT-PCR, Western blot, IFA detection, and nalysis of genic stability certified that genetically stable rAd-Cap-InvC can effectively mediate Cap-InvC fusion protein. The titers of rAd-Cap-InvC and rAd-Cap can reach 109.32 TCID50/mL.Second, the animal immune experiment showed that the recombinant adnovirus could mediate laboratorial bodies producing PCV2 specific antibodies effectively. Both rAd-Cap-InvC and rAd-Cap was injected into Kunming mice as 108TCID50 a mouse. And repeat once under the same condition 14 days later. PCV2 specific antibody was detected at 14 d, 21 d, 35 d, and 42 d after the first injection. The results showd that no matter rAd-Cap-InvC or rAd-Cap injected, the mice producted PCV2 specific antibody. But the amount of the antibody of rAd-Cap-InvC injected mice was higher than rAd-Cap injected mice, which existed in whole experiment time quantum significantly(P<0.05).Recombinant adnovirus rAd-Cap-InvC was injected into two groups of piglets as 1010TCID50 a body(large-dose group) and 109TCID50 a body(nomal-dose group). Recombinant adnovirus rAd-Cap was injected into one group of piglets, and rAd for one group. Each group owned 2 piglets. Repeat injection under the same condition 14 days later. PCV2 specific antibody of piglets was detected at 14 d, 21 d, 28 d, and 36 d after the first injection. The results showd that the antibody titer of large-dose rAd-Cap-InvC group is higher than the titer of nomal-dose rAd-Cap group. The antibody titers of nomal-dose rAd-Cap-InvC group is higher than rAd-Cap group`s.Animal immune experiments showed that both recombinant adnovirus rAd-Cap-InvC and rAd-Cap could stimulate injected bodies producing PCV2 specific antibody. And the rAd-Cap-InvC`s is more.This research constructed the recombinant adnovirus co-expressing PCV2 Cap protein and immunological enhancing factor InvC, which could stimulate injected animals producing more PCV2 specific antibody. InvC promoting antibody production is deduced.