Cloning And Expression Of Riemerella Anatipestifer OmpA With Duck Interleukin-2 Fusion Gene

Riemerella anatipestifer (RA) infection disease is an acute or chronic diseases caused by RA. This infection disease can bring about most of poultries and wild birds such as domestic ducks, geese and turkeys appearing pericardia fibrin parahepatitis, pericardia fibrin exudation, pericardia fibrin meleagridis, meningitis, salpingitis, arthritis and other clinical manifestations. It accounts for significant economic losses due to high morbidity and mortality. The disease is prone to mixing infection with other pathogenic microorganisms, for example avian E.coli, salmonella, streptococcus, chlamydia, avian pasteurella and so on. Once ducks or goose flocks infected this disease, it becomes endemic easily and is hard to eradicate, with repeated infectious episodes possible. To date,21 serotypes RA have been reported, and some new serotypes kept on appearing. Currently, drug therapy is a main treatment measure for this disease. However, it can cause resistant RA strains appeare and effect food safety. Therefore, immunization is the key of prevention of this disease. In recent years, the researchers focused on subunit vaccine on high protective antigens of RA. Outer membrane proteins (OmpA) of RA were acknowledged to be highly protective antigens.Interleukin-2 (IL-2) is a kind of important cytokines produced by helper T cells and NK cells after suffering mitogen or antigen stimulation .IL-2 is the core of immune regulation and achieves the adjustment of the body's immune function with other cell factors. IL-2 can not only stimulate T cells, B cells and NK cells differentiation, proliferation and secreted antibody, but also promote immune adjustment factors to release, including interferon and tumor necrosis factor. Therefore, IL-2 is one of the most important factors to guarante the normal immune function. Poultry IL-2 can not be used on human and other mammals due to IL-2 have certain species specificity. Currently, there are some reports on gene engineering vaccine related to IL-2 of chicken and goose to prevent some infectious diseases, but the application of duck IL-2 is rare. To pave the way for new vaccine exploration and development, this study will apply IL-2 on OmpA genetic engineering vaccine of RA to incease duck RA vaccine immunologic.A gram-negative bacterium had been isolated from infective ducks' liver, brain and heart blood. All The results of morphological observation, physical and chemical identification assays, and pathological and histological observation conformed to the features of RA. Compared the homology of the strain JL-RA1 (bankit HQ392516) 16S rRNA gene sequences with the reported RA is up to 99.1% to 99.9%. For example, the homology of JL-RA1 compare to RA ATCC11845 (bankit U60101), RAf54 (bankit EU016547) and RA D-24046(bankit EU871818) are 99.6%,99.9% and 99.9%, respectively. The phylogenetic analysis of JL-RA1 showed that JL-RA1 with RA-YM (bankit FJ031240), RA f75 (bankit EF641572), RA f54 (bankit EU016547) and RA D-24046 (bankit EU871818) is closer but distant to RA H-2565 (bankit AY871820).PCR amplify the 1 182bp OmpA gene by specific primers with a restrict enzyme site in the upstream and a complementary linker in the downstream from RA strain JL-RA1. Extracted total RNA from healthy adult ducks peripheral blood lymphocytes stimulated by ConA, and then designed of specific primers with a complementary to the upstream linker and a restriction site in the downstream to amplify 381bp DuIL-2 mature protein gene by RT-PCR. We successful got the amplification of 1 551bp OmpA-DuIL-2 fusion gene by SOE-PCR with OmpA forward primer and DuIL-2 revease primer. The fusion gene was ligased to E. coli expression vector pET-28a to construct a pET-OmpA-DuIL-2 fusion expression vector, and then transformed the expression vector into E. coli Rosetta (DE3). The DE3 contained pET-OmpA-DuIL-2 fusion expression vector can express target proteins by IPTG induction. The SDS-PAGE results showed that the recombnant protein molecular weight is about 61KD, and Western-Blot showed the protein owning both OmpA and DulL-2 immunogenicity.