Immune-enhancement Of Taishan Pinus Massoniana Pollen Polysaccharides On Recombinant Porcine Reproductive And Respiratory Syndrome Virus Glycoprotein 5 Protein

Porcine reproductive and respiratory syndrome(PRRS) is a highly contagious viral disease, it has a high disease incidence and mortality. It is susceptible to pregnant sows and piglets within 1 month old, and it mainly causes porcine respiratory distress and reproductive disorders, which has brought great economic losses in commercial flocks. PRRS mainly focusing on prevention, the live attenuated vaccine and inactivated vaccine were used as commercialization vaccine. The live-attenuated PRRSV vaccines exhibited the risk of viral spread, and the inactivated vaccines only induce a limited protection. Therefore, it is crucial for developing a novel subunit vaccine against PRRS infection. In addition, the selection of an effective adjuvant is significant to improve the immune response of subunit vaccine. Herein,we expressed GP5 through prokaryotic expression system to prepare a subunit vaccine, and then to evaluated its immune effects. Taishan Pinus massoniana pollen polysaccharide(TPPPS) was used as adjuvant to investigate the immunomodulation effects.The identification and expression of recombinant plasmid of PRRSV GP5.To identify the recombinant plasmid PET-32a-GP5 via PCR and double digestion, and sequenced to verify the correct recombinant plasmid PET-32a-GP5 was transformed into BL21(DE3) pLysS competent cells, then express the prokaryotic proteins. We can obtain a target band that size of about 32 KD by SDS-PAGE analysis of the expressed protein. Meanwhile, we can obtain a specific imprinting size of 32 KD by Western-Blot which is consistent with SDS-PAGE analysis. At last, the obtained protein was purified by nickel column His tag.Immunopotentiation of Taishan Pinus Massoniana Pollen Polysaccharides on the immune responses induced by recombinant Porcine Reproductive and Respiratory Syndrome Virus GP5 Subunit Vaccine. In this study, 72 SPF Kunming mice(weight of about 20 g) were divided into 6 groups(I-VI) randomly. Each mouse in group I-VI was vaccinated with pure GP5 subunit vaccine, 20, 40, and 60 mg/mL of TPPPS adjuvant GP5 subunit vaccines, the Freund's incomplete adjuvant GP5 subunit vaccine, and PBS respectively. The secondary vaccination was conducted at 7 days post the first vaccination. The titers of serum antibody,percentages of T lymphocyte, lymphocyte transformation rate, and concentrations of cytokines were tested by ELISA, flow cytometry, MTT, and ELISA kits, respectively, at one week interval. The results showed that all immune indexes of mice from groups II- VI have a significant improvement than those of group I. Furthermore, all immune indexes in Freund's adjuvant group and TPPPS adjuvant group were higher than the pure GP5 group. Among three concentrations of TPPPS, 60 mg/mL TPPPS showed optimal effects. Our results demonstrated that the recombinant PRRSV GP5 exhibited immunogenicity, and TPPPSsignificantly enhanced the immune responses induced by GP5 subunit vaccine.