Secreted Expression Of Porcine Interferon Alpha In Picha Pastoris And Its Vaccine Adjuvent Fuction Investigation

In our country, we have the largest pig-farming industry in the whole world. At present, there are many infectious diseases that cumber the thriving and the updating of this industry. So there are urgent wishes and needs to develop the new generation of the safe and truly effective antiviral drugs and immune potentiator or adjuvant to enhance the health of pigs and improve the efficacy of the current vaccine. Interferons are cytokines that function as antiviral factors and immune regulators, which are naturally produced by the immune system of mammal animals immediately following viral infection or viral vaccination. The interferon system is the first frontier of defense against viral infection in mammals. Interferon are classified into two types, namely Typeâ… and Typeâ…¡interferon, according to the diversity of amino acid sequence, the antigenicity and the type of cells from which they are produced. Interferon alpha and interferon beta are the major members of typeâ… interferon and the only one of typeâ…¡interferon is interferon gamma. Typeâ… interferon is famous as the potent antiviral factors, while interferon gamma were manifested as the stronger regulators for immune and antiviral potent factors with only a little lower potency than Typeâ… interferon. The Recombinant human interferon had been successfully used to cure the viral diseases and tumor since decades ago. Recent years, the porcine interferon's antiviral function and the adjuvant potency for vaccine have been found and conformed through many experiments and trials. One aim of this experiment is developing the recombinant porcine interferon alpha, with gene and protein engineering technique, of which we have the fully independent intellectual property right. Another aim of the present study is proving their antiviral potency, in order to pave the way for the production of the engineered porcine interferon in the large scale and their application in pig farming industry.This study includes: Porcine interferon alpha mature protein encoding gene was sub-cloned from pGEM-T-A and then inserted into pPICZαA vector and result in pPICZαA-PoIFNA. The recombinant plasmid was linearized with SacI and was then transformed into Pichia pastoris X-33 strain by electroporation. The existing of PoIFN-αgene in the transformed yeast cells was detected by PCR using PoIFN-αprimers. Weather the PoIFN-αgene was integrated into the alcohol oxidase promotor (AOX1) locus on yeast chromosome was verified by amplification with AOX1 primers. The identified transformants were isolated and assayed for the expression of PoIFN-α. The expression of PoIFN-αwas induced with methanol in BMMY culture medium. The existing of PoIFN-αin the supernatant of culture was firstly detected through SDS-PAGE, and then the positive samples were further identified through antiviral activity testing. As a result, the expressed poIFN-α₁ is verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in MDBK cells, which is about 2x10⁶U/mL.In the present study, the antiviral activity of yeast expressed porcine interferon alpha (yPoIFN-α) was evaluated by testing the inhibition effect on VSV in different cells such as MDBK, PK-15 and Marc-145. The sensitivity of different strains of PRRSV and PRV to yPoIFN-αwas also surveyed. The result indicated that the anti-VSV activitie of yPoIFN-αwas about 10^6.5U/mL in MDBK, which is100 times higher than in PK-15 and Marc-145. The mechanism underlined maybe different level of interferon receptor in these cell lines. The high pathogenic strain and nonpathogenic strain of PRV are both sensitive to yPoIFN-αand the virus replication in 1000U/mL yPoIFN-αpretreated PK-15 was about 100 times lower than that in control cell. PRRSV replication in yPoIFN-αpretreated Marc-145 was also inhibited, two strains of PRRSV, PRRSV VR2332 and PRRSV JS06 a field isolated strain from Jiangsu province are the same sensitivity to yPoIFN-α. In conclusion, the anti-viral activity of yPoIFN-αon PRRSV and PRV was demonstrated clearly in the present study.The immunogenicity of porcine circovirus type 2 (PCV-2) Cap protein Secreted expressed by Pichia Pastoris and adjuvant effect of recombinant porcine interferon alpha (rPoIFN-α) were examined in piglets in the present experiment. Groups of piglets were inoculated with recombinant Cap protein in the presence or absence of rPoIFN-αin a prime-boost protocol. Then the piglets were challenged with PCV-2 JS strain at 14 days post the boost-vaccination. Neutralizing antibody against PCV2 was produced in piglets inoculated with Cap protein of PCV2 expressed by Pichia Pastoris. The use of rPoIFN-αresulted in significantly higher ELISA antibody and neutralizing antibody levels on recombinant Cap protein inoculation. After challenge all control piglets developed viremia and had a long time fever, only one of four piglets receiving recombinant Cap protein of PCV2 developed viremia, while addition of rPoIFN-αproduced an improved protection to the viral challenge, since PCV2 replication was completely inhibited. As evaluated by viremia ratio, clinical signs (fever), growth parameters, the piglets were protected against PCV2 challenge after vaccination of rCap. rPoIFN-αshowed potential adjuvant effects on recombinant Cap protein of PCV-2, which was characterized not only in antigen specific humoral immune response, but also in protection against PCV-2 infection.