Study On Construction And Immunogenictiy Of Recombinant Adenovirus Expressing G Protein Of Vesicular Stomatitis Virus

Vesicular stomatitis(VS) is an acute highly contagious human zoonotic infectious disease caused by the vesicular stomatitis virus(VSV). It was classified as a notification disease by OIE, and it is one of the ClassⅡanimal diseases in China. Now, there is no effective treatment for the disease and it can only be prevented and controlled by the vaccine. Some researches of vesicular stomatitis virus vaccines have been done at home and abroad, but the research of new vaccines needs further development.VSV glycoprotein(G) positions on the protrusions of the envelope and stimulates the production of neutralizing antibodies, as well as presenting the specific type strains, is the preferred antigen for VSV engineered vaccine research. In this study, we conducted the recombinant adenovirus expressed two serotypes G proteins and the fusion protein of VSV G protein by a human type 5 replication-defective adenovirus expression vector and conducted the preliminary analysis of their immunogenicity. Three paris of primers were designed to amplify the VSV-IN-G、VSV-NJ-G and VSV-IN-NJ-G genes. We amplified the VSV-IN-G gene and VSV-NJ-G gene by PCR and amplified the VSV-IN-NJ-G gene by overlapping PCR with the polypeptides GlyGlyGlyGlySer.The three genes were cloned into the adenovirus shuttle vector pacAd-CMV K-NpA to construct the recombinant shuttle plasmids containing the target genes.The recombinant shuttle plasmids were identified by PCR,restriction and sequencing. The recombinant shuttle plasmids and adenovirus backbone plasmid linearized with PacⅠrestriction enzyme were cotransfected to the AAV-293 cells and the three kinds of recombinant adenovirus rAd-VSV-IN-G,rAd-VSV-NJ-G and rAd-VSV-IN-G-NJ-G were packaged successfully. The three kinds of recombinant adenovirus inoculated into the AAV-293 cells were serially propagated to 20 generations and the virus titers were stable by determineing the TCID50. By indirect immunofluorescence and western blot detection method for detecting the expression of the target proteins, it showed that G proteins can be expressed and showed good reactogenicity.In order to evaluate the immune affection of the recombinant adenovirus, the study selected 50、 6-8-week-old female BALB / c mice, which were randomLy divided into five groups, each group of 10. The mice were respectively inoculated subcutaneously three times at 2-week intervals with recombinant adenovirus.After the first inoculation 0, 2, 4, 6 weeks collected serum to detect the specific antibody levels by indirect ELISA. The results showed that the three groups also can induce the production of VSV-specific antibodies. In virus neutralization test for detection of neutralizing antibody level, the antibody levels were 1:16 to 1: 32 of three groups. The results of Lymphocyte proliferation test to detect levels of immune cells show that the vaccines can cause a strong immune mouse lymphocyte proliferation, and are significantly higher than the negative control group. These results showed that the three kinds of recombinant adenovirus can express foreign proteins very well, and the proteins can cause a certain degree of humoral and cellular immune responses in immunized mice, laid a foundation for further studies of the recombinant adenovirus vaccines expressing VSV glycoprotein.