Construction Of Attenuated Salmonella Typhimurium Strain Expressing Cap Protein Of PCV2 And Animals Immunization

This study aimed to construct a kind of live vector vaccine which could express Cap protein of PCV2 by attenuated Salmonella typhimurium(S.t), and evaluated the live vector vaccine by animals immunization. Capsid protein was encoded by ORF2 gene of PCV2, and this protein has been identified as the major structural and immunogenic antigen of PCV2. Therefore, the ORF2 gene deleted the 41 amino acid was chosen as an aimed gene in this study. Utilizing the balanced lethal symtem of attenuated Salmonella typhimurium to express the optimization of the ORF2 gene, construct live vector vaccine expressed Cap protein, and to evaluate biological traits and immunological properties of the vaccine strain. Some results were as follows:1 Construction of attenuated S.t live vector vaccine expressing Cap protein of PCV2Specificity primers P1 and P2 was synthesized and the optimization of the ORF2 gene was amplified, then the ORF2 gene was inserted into the attenuated S.t expression vector pYA3341 containing asd gene by double digestion of BamH I and Pst I, and constructed the recombinant expression plasmid pYA3341-ORF2.The reconstructed plasmid was electrotransformed into a balanced-lethal recombinant attenuated S.t strain X3730 and X4550 with one by one, which were deltd aspartate-β-semialdehyde dehydrogenase (ΔAsd), adenylyl cyclase (ΔCya) and cyclic AMP receptor protein gene (ΔCrp). In the end, we got oral vaccine strain X4550 (pYA3341-ORF2).2 Detection of the biological characteristics of recombinant vaccine strainThe vitro stablity, growth characteristic, orally safety of recombinant vaccine strain X4550(pYA3341-ORF2), and distribution in mice after orally administered of the recombinant vaccine were also analyzed. The results showed that the Cap protein in the X4550 had a high level of expression, and the recombinant strain had good genetic stability in vitro, growth states, safty, non-toxic and well distribution in the body.3 Research of the immunogenicity of recombinant vaccine strain Treatment group was immunized intragastrically with recombinant vaccine strain X4550 (pYA3341-ORF2), negative control group were immunized intragastrically with recombinant strain X4550 (pYA3341) and control group were taken orally with PBS. Antibody titer of serum of treated mice can reach to 1:1920 by ELISA; Neutralization activity of the treated mice serum can reach to 1:19.2 by indirect immunofluorescence; Stimulation index of lymphocyte of the treated group was 4.26 by MTT; The CD4+ and CD8+ lymphocyte subsets populations of immuned mice were separately 29.9, 7.52 by flow cytometry, and the ratio of CD4+/CD8+ was 3.99. Then the piglets were also immunized intragastrically with recombinant vaccine strain X4550 (pYA3341-ORF2), negative control group were immunized intragastrically with recombinant strain X4550 (pYA3341) and control group were taken orally with PBS. Antibody titer of serum of treated piglets was 1:57.6 by ELISA; Neutralization activity of the serum was 1:11.2 by IFA; Stimulation index of lymphocyte of the treated group was 3.82 by MTT.Above studies suggested that the recombinant vaccine strain can express stably Cap protein of PCV2, which had good immunogenicity. This would supply some dates for further develop of vaccine and clinical application in the nearly future.