Expression Of Porcine Interferon-a And Interferon-γ Using Baculovirus/insect Cell System And Preliminary Application Of Its Recombinant Protein

1: Clone of Porcine Interferon-αand Interferon-γGenesTwo pairs of RT-PCR primers were designed and synthesized based on the published gene sequence of porcine interferon-α(PoIFN-α)and interferon-γ(PoIFN-γ).The two about 500bp target DNA sequences were amplified by reverse transcription polymerase chain reaction depending on the template of total RNA which isolated from ConA-stimulated porcine spleen cell,and cloned into pGEM-T vector.The two gene fragment were identified to be interferon-αand interferon-γwith the methods of restriction enzyme digestion,polymerase chain reaction,and sequencing.Comparing with the sequences of porcine that deposited in NCBI.There are completely consensus with these. That will allow us to explore this cytokine as a poteintial therapeutic agent for porcine diseases using a baculovirus/insect cell expression system.2: Effects of the honeybee melittin signal peptide on expression of porcine interferon-αin insect cellsThe baculovirus-insect cell system is a valuable tool for the expression of heterologous proteins. Due to limitations in the intracellular processing environment, however, heterologous secreted and membrane proteins are often insoluble.Many attempts to modify the insect cell secretory pathway by overexpressing processing factors have demonstrated the potential to overcome these limitations.Report by Tessier and his co-worker showing that secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptede: the honeybee melittin signal peptide(HBM).However, There are studies showed that insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. The aim of this study was to investigate the effects of the honeybee melittin signal peptide on baculovirus-mediated expression and secretion of porcine interferon-α.The HBM gene was amplified by PCR using a pair of primers with partial overlapping sequence.The synthetic chimaera was obtained by ligation the sequence coding mature porcine interferon-αwith HBM by PCR.The other fragment without HBM was amplificated also.Each gene was cloned into the baculovirus transfer vector pFastBacTMDual,under the control of pH promoter. Constructs were transformed into DH10BacTM E.coli,the site-specific transposition occurs between the mini-Tn7 element on the pFastBacTMDual vector and the mini-attTn7 target site on the bacmid to generate a recombinant bacmid ,then introduced to Spodoptera frugiperda.Immunofluorescence assay and western blot analysis confirmed the secretory expression of recombinant baculovirus with HBM.Overxpression of the porcine interferon-αform has also been achieved using recombinant baculovirus without HBM but mostly in an aggregated form with no secretion. Using the honeybee melittin signal peptides in baculovirus expression vectors is enable to aid in increasing expression and yield of heterologous secreted proteins in insect cells.3: Secretory Expression of Porcine Interferon-γusing a baculovirus system and its antiviral activity detectionCytokines, such as porcine interferon-gamma(PoIFN-γ) have been shown to have antiviral and adjuvant activity in livestock and have the potential to be used as alternatives to antibiotics. This part aimed to the expression of PoIFN-γin a baculovirus system to generate a fully functional recombinant protein. Porcine interferon-γcDNA was cloned from mitogen stimulated spleen lymphocytic total RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The sequences encoding for a mature 145 amino acid protein in frame with a C-terminal 6×Histidine tag,was cloned and expressed into baculovirus transfer vector pFastBacTMDual,under the control of pH promoter.The authentic signal sequences of porcine interferon-γwere substituted with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell.The recombinant proteins were successfully detected in expressing cells by immunofluorescence assay and in the culture medium by western blot analysis.The recombinant PoIFN-γis verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in PK-15 cells, which is about 6.67 x 10(5) U/mL in supernatant.4: Co-expression of porcine interferon-α&γusing a baculovirus system and its antivirus activity detectionPorcine interferon(interferon,IFN) comprises IFN-α, IFN-βand IFN-γ.Type I IFNs (IFN-αand IFN-β) and type II IFN (IFN-γ) are important components of the host immune response to viral infections. IFN-αand IFN-βare produced by most cells as a direct response to viral infection, while IFN-γis synthesized almost exclusively by activated NK cells and activated T-cells in response to virus-infected cells.Like type I IFNs,IFN-γhas antiviral activity,but in addition it also posses immunomodulatory activity. Combination of type I and type II interferons act synergistically such a mixture of the two has much greater activity than that expected from the separate contribution of each type. Baculovirus/insect cell system was used for such mixture recombinant proteins production.Porcine interferon-αgene and interferon-γgene were cloned into pFastBacTMDual vector that has two promoters and cloning sites,allowing simultaneous expression of these two genes.The two proteins in frame with a C-terminal hexahistidine(His6) tag to facilitate purification of the secreted protein on nickel-containing resins. The authentic signal sequences of the two genes were substituted with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell.Both genes were cloned into the baculovirus transfer vector pFastBacTMDual,under control of pH promoter and p10 promoter,respectively.A recombinant baculovirus,Bac-PoIFN-α&γ, involving two heterogenous genes, was generated by transfecting pBac-PoIFN-α&γto bacmid inside DH10Bac Escherichia coli by site-specific transposition,followed by transfection into the sf9 cells.The expressed recombinant glycoproteins were successfully detected in expressing cells by SDS-PAGE and by western blot analysis. Secretory expression of the recombinant protein was verified by immunofluorescence assay that indicating immunofluorescence signal mainly locate at cytomembrane.The bioactivity was confirmed to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in PK-15 cells, which is about 3.24×107 U/mL in supernatant and 5.86×106 U/mL in insect cells, respectively.5: Inhibition of porcine reproductive and respiratory syndrome virus in Marc-145 cells by compound of recombinant porcine interferon.Porcine reproductive and respiratory syndrome (PRRS) is caused by an enveloped positive-stranded RNA virus placed in the family Arteriviridae,Infection of adult pigs generally produces a nonfatal disease characterized by flu-like symptoms, including mild interstitial pneumonia, elevated temperature, and inappetance. In sharp contrast, the infection of pregnant gilts and sows results in abortion and the birth of dead and weak-born piglets.Surviving piglets exhibit the severest form of respiratory distress with mortality often approaching 100% within 3 weeks after farrowing. Surviving pigs continue to suffer the negative effect of PRRSV by exhibiting increased susceptibility to secondary infection. Pigs surviving acute PRRS support a long-term asymptomatic infection, which has contributed to the worldwide spread of the disease. The persistent infections result large economics loss in pig raising.Rowland reported that pretreatment of MARC-145 cells with IFN-γinhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-γon virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells.The purpose of this part is to investigate the inhibited effect of recombinant compound IFN on PRRSv in Marc-145 cells with the cytopathic effect.The result show that the dilution of 210 of crude recombinant supernant can protect the Marc-145 from the cytopathic effect cause by 100TCID50 of Porcine reproductive and respiratory syndrome virus.