Recombinant Lactobacillus plantarum expressing HA2 antigen elicits protective immunity against H9N2 avian influenza virus in chickens

Low pathogenic H9N2 subtype avian influenza virus (AIV) can lead to moderate respiratory symptoms and low egg production rates in poultry. Due to its immunologic suppression, other various infectious pathogens give rise to the co-infection of hosts, causing heavy economic losses in the commercial poultry industry in both China and worldwide. Therefore, it is time to explore a novel, safe, and effective vaccine. We have already made use of the surface of Lactobacillus plantarum to display AIV HA2 (NC8-pSIP409-pgsA′-HA2), which demonstrated that it has a good immunogenicity. In this study, by evaluating the immune protection effect of NC8-pSIP409-pgsA′-HA2 on chickens, we found that the hemagglutination inhibition (HI) antibodies, specificity IgG antibody in chickens, the sIgA titer in broncho alveolar lavage fluids (BALF), and the T cell response were increased notably after oral vaccination with NC8-pSIP409-pgsA′-HA2. In addition, weight loss, lung titers, and lung pathologies were improved when chickens were orally vaccinated with NC8-pSIP409-pgsA′-HA2 after challenge with H9N2 AIV. This strategy lays the foundation for the development of recombinant L. plantarum oral vaccines in the prevention of AIV.

     

Expression and purification of swine RAG2 in E. coli for production of porcine RAG2 polyclonal antibodies

Recombination activating gene 2 (RAG2) is necessary for immature B cell differentiation. Antibodies to human and rabbit RAG2 are currently commercially available, but antibodies to swine RAG remain unavailable to date. In this study, the swine RAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Tobacco Etch Virus protease. The cleaved protein was purified by molecular-exclusion chromatography and named pRAG2. We used pRAG2 to immunize rabbits, collected the serum and purified rabbit anti-pRAG2 polyclonal antibodies. The rabbit anti-pRAG2 polyclonal antibodies were tested via immunofluorescence on eukaryotic cells overexpressing pRAG2 and also able to recognize pig natural RAG2 and human RAG2 protein in western blotting. These results indicated that the prepared rabbit anti-pRAG2 polyclonal antibodies may serve as a tool to detect immature B cell differentiation of swine.