Study On Expression Of Porcine Interleukin-2 & Porcine Interleukin-6 And Their Adjuvant Effects On Vaccines

In pork-producing community, various infectious diseases which are caused by viral or bacterial pathogens, badly restricted the development of natural and regional farming industries, and also seriously affect human health in whole world. Hence, it's necessary and great meaningful in both economic development and societal stabilization to prevent and control infectious diseases. Since now vaccination is still the most effective and economical means for the aim. But current routine vaccines are shown not good enough to defense pathogen infection because of the shortages on efficiency and safety. Hence, how to improve the efficiency of vaccine under a safe dosage, become one of the hottest topics in vaccine research area.Cytokine, produced by immune cells and other correlative cells, is a kind of pleiotropic protein which plays important roles during induction and modulation various immune responses. It's cytokine's bioactivity in immune system that inspires people to use it as adjuvant to improve vaccine efficiency. After beyond 20yr's research, scholar in whole world had devoted themselves in exploring the adjuvant effects of cytokine on vaccine and got many excited achievements. They found various cytokines, including Interleukins, Interferon, TNF, GM-CSF et al, could enhance more or less the efficiency of different routine vaccine such as inactivated vaccine, attenuated vaccine and subunit vaccine. Recently, research in cytokine genes as DNA vaccine adjuvants, became popular and absorbed more and more attention because of its construct conveniently and function effectively.In this study, porcine interleukin-2 (pIL-2) and porcine interleukin-6 (pIL-6) were used to explore their adjuvant effects on routine vaccine (PRV inactivated vaccine) with the form of recombinant proteins, and on DNA vaccine (PRRSV ORF5 DNA vaccine) with the form of gene. The main research included as below: 1. Expression of pIL-2, pIL-6 and their fusion protein pIL6-IL2 in E. coli andpurificationConstructed three prokaryotic expression plasmids pET-IL2, pGEX-IL6, 28a-IL6-IL2, and transferred into E. coli BL21 (DE3) expression strain. Three insoluble recombinant proteins were produced. Among them, rpIL-2 was included its signal peptide, rpIL-6 was designed to cut off signal peptide for expression successfully, rpIL6-IL2 was linked by a fragment which consisted of hydrophilic and low charge base pairs. The method contained with inclusion body collection — SKL denaturalization — dialysis re-naturalization was used to purified these three prokaryotic expression products. Also,purified rpIL-2 and rpIL-6 were used to immune rabbit for specific rpIL-2/rpIL-6 antibody generation2. Expression of pIL-2n pIL-6 and their fusion protein pIL6-IL2 in P. pastoris and purificationConstructed three P. pastoris expression plasmids pPIC3.5K-IL2, pPIC9K-IL6, pPIC9K-IL6-IL2, and transferred into P. pastoris GS115 strain. Three recombinant proteins were secreted into the culture medium with expected molecular weights after the recombinant P. p strains were induced by 1% methanol. All three P. pastoris products were analyzed by Western blot, treated with PNGase F and purified through (NH4)2SO4 deposition — PBS dialysis — PEG20000 concentration to eliminate the toxic in cultural medium. The PNGase F treatment results show that P. pastoris product rpIL-2 contained approximately 5kDa N-linked glycosylation, rpIL-6 hadn't N-linked glycosylation, and a mixture of the N-linked glycosylated (about 2kDa) and nonglycosylated forms was appeared in rpIL6-IL2. Three recombinant P. pastoris strains were analyzed the best induction time and the stabilization for foreign protein expression. The results show 4th or 5th day was the best induction time, and recombinant yeast cells could grow and secret foreign protein stably only if cultural content is proper. Lyopholozed purified P. pastoris products were stable in 4°C for 4 months.3. Identification and comparison of bio-activities of recombinant proteins in two expression systemsThe biological activities of three recombinant cytokine expressed in E. coli and P. pastoris systems were determined by cell proliferation assays using IL2-dependent cell line CTLL-2 and IL6-dependent cell line B9. The results show the bio-activities of P. pastoris products were higher approximately 10-100 times than E. coli products. Then three P. pastoris recombinant cytokines were used in following animal test.4. Pig assay to explore the adjuvant effects of three recombinant cytokines on PRV inactivated vaccine.Pigs were injected 2mL of PRV inactivated vaccine once and inoculated l*105 U candidate cytokine adjuvant three times. 8 weeks after immunization, all pigs were challenged by high dosage PRV (Ea) strain. The results from VN tests and CTL assays showed that among three recombinant cytokines, rpIL-2 could furthest enhance PRV-specific humoral and cellular immune responses. Moreover, pigs of PRV+IL2 group got a significant higher protection than ones of control group after PRV infection. Unfortunately, we did not observe obvious positive effect of rpIL-6 and rpIL6-IL2 administration on PRV IAV vaccination, in both immune responses and protection ability.5. Construction of DNA vaccines containing PRRSV ORF5 gene and different cytokine gene in two expression cassettes.Constructed 4 eukaryotic expression plasmids containing two expression cassettes: pCI-ORF5-neo, pCI-ORF5-IL2, pCI-ORF5-IL6 and pCI-ORF5-IL6-IL2, and then transfected into Hela cell. IL2-dependent cell line CTLL-2 and IL6-dependent cell line B9 were used to identify whether the foreign cytokine had been expressed in this mammalian cell.6. Mouse assay to explore the adjuvant effects of three recombinant cytokine genes on PRRSV ORF5 DNA vaccine.Mice were immunized twice and sampled at 4, 6 and 8 weeks for analyzing antibody levels by ELISA and VN tests. All three interleukin genes showed adjuvant efficiencies on PRRSV ORF5 DNA vaccines by enhancing mice humoral immune responses.Adjuvant efficiencies of porcine cytokine proterins in PRV inactivated vaccine were tested in this study, aimed to obtain an easy-prepared and highly potent cytokine adjuvant to improve the efficiency of PRV inactivated vaccine and other routine vaccines, defense and control infectious disease outbreak, promote pork-producing industry development. Moreover, in order to enhance DNA vaccines efficiency, develop a new potent DNA vaccine adjuvant, adjuvant efficiencies of porcine cytokine genes in PRRSV ORF5 DNA vaccine were explored in this study.