The Functional And Structural Studies On A Novel Recombinant Porcine Interferon-α

Interferon is a type Ⅱ cytokine which possesses many biological functions including immunomodulation, antiviral activities and antiproliferative activity. According to difference in receptor, chromosomal localization and acid stability, interferon can be classified into three types I, II and III. IFN-a belongs to type I interferon, which has powerful antiviral activity. In order to study the biological activity of interferon and expand their clinical application, we designed and expressed a consensus porcine IFN-a (CoPoIFN-a), and studied its structure and functions. The purpose of the experiments is to design a recombinant consensus porcine IFN-a and explore its safety and effectivity to produce a novel interferon-a to increase pig immunity and the protective capacity of current vaccines. We also screened the crystal growth conditions of CoPoIFN-a and modeled the ternary structure of the IFN-as with its receptors in order to determine the interactions between interferon and the receptors, which will help us to study the activity of interferons and design the novel interferions with higher biological activities.1. The design, gene synthesis and cloning of CoPoIFN-aWe searched protein sequences of17native porcine IFN-a subtypes from NCBI, and aligned the sequences using Bioedit and assigned the most frequently observed amino acid in each corresponding position. The designed interferon-α was named as CoPoIFN-a. Based on codon usage of yeast, the nucleotide sequence of CoPoIFN-a was designed, and14primers were designed and synthesized to amplify CoPoIFN-a gene using gene splicing by overlap extension PCR (SOE-PCR) method. The full length CoPoIFN-α gene was then cloned into pPICZa expression vector using an upstream EcoRl site and a downstream XbaIsite. The positive Pichia pastoris transformant carrying CoPoIFN-a gene were confirmed by PCR and DNA sequencing. 2. The expression and purification of CoPoIFN-αThe Pichia pastoris X-33strain carrying CoPoIFN-a or control PoIFN-a gene was induced with methanol under28℃for72h. The supernatant was separated from the cells by centrifuging at12000r/min under4℃. The supernatant was first precipitated with30%(NH4)2SO4to remove some contaminant proteins, and the fraction containing IFN was obtained by precipitate with45%(NH4)2SO4. Further purification steps were carried out by using hydrophobic, ion exchange and gel filtration chromatography. The purified proteins were confirmed by using mouse anti-porcine IFN-al monoclonal antibody. The secondary structure of purified CoPoIFN-α and PoIFN-a were analyzed by circular dichroism spectroscopy method, the data showed they exists mainly as α-helixes in solution.3. The in vitro biological studies on CoPoIFN-αWe assayed biological activities of CoPoIFN-a, and compared the differences between CoPoIFN-a and PoIFN-a. First of all, we compared their inhibitory abilities on the VSV infection in MDBK, PK-15and MARC-145cells. We observed that the antiviral activity of CoPoIFN-α was46.4,63.6,53.5-fold higher than that of PoIFN-a in MDBK, PK-15and MARC-145cells, respectively. We next compared the ability of CoPoIFN-a or PoIFN-a in inhibiting PRV and PRRSV replication in the cells that have been pretreated with CoPoIFN-a or PoIFN-a. We found that the viral titers of PRV in PK-15cells pretreated with PoIFN-a were higher than that pretreated with CoPoIFN-a by25-fold, and real-time PCR result showed that the amount of PRV mRNA in PK-15cells pretreated with PoIFN-a was4.8-fold higher than that pretreated with CoPoIFN-a. The results on PRRSV indicated that the viral titers of PRRSV in MARC-145cells pretreated with PoIFN-a were higher than that pretreated with CoPoIFN-a by10-fold, and real-time PCR result showed that the amount of PRRSV mRNA in MARC-145cells pretreated with PoIFN-a was5-fold higher than that pretreated with CoPoIFN-a. The antiproliferative activity of IFN-as against PK-15cells was investigated in vitro using the MTT assay after treating the cells with serial dilution of8-0μg/mL CoPoIFN-a or PoIFN-a, the results showed the antiproliferative activity of CoPoIFN-a was higher than that of PoIFN-a within a concentration range of0.25-0.5μg/mL, and significantly higher within a concentration range of1-8μg/mL. Finally, we assayed the stimulation ability of the CoPoIFN-a or PoIFN-a on Mxl, OAS1and PKR gene induction. The Q-PCR results showed the mRNA levels of Mx1and OAS1 gene in the PK-15cells were significantly enhanced. In the CoPoIFN-a treatment group, the mRNA level of OAS1was enhanced by about3.2-fold compared with PoIFN-α treatment group, while the level of Mxl was increased by about4.6-fold. We did not detect PKR mRNA expression in PK-15cells.4. The studies of adjuvant effect of CoPoIFN-a in CSFV peptide vaccineTo assess the adjuvant effect of CoPoIFN-a in CSFV peptide vaccine, we ligated a gene corresponding to the peptide (aa693-716) from CSVF envelope protein E2downstream of trxA gene in the pET-32a vector. We purified the fusion peptide from E. coli. from inclusion body. The fusion peptide was emulsified with206adjuvant to generate CSFV peptide vaccine pb. The30-day old pigs were immunized with pb combined with104unit,105unit or106unit CoPoIFN-a and boosterd two weeks later. The peripheral blood was colleted each week before and after the first immunization. The CSFV antibody in the blood was detected using ELISA. Two weeks after the first immunization, we carried out antibody neutralizing test. We also seperated peripheral blood lymphocyte and stimulated the cells with fusion peptide antigen to test their proliferative and IL-4, IFN-y secretion ablities. The results showed pb peptide vaccine can induce the humoral immune reaction in pigs, and CoPoIFN-α can increase the reaction level in dose-dependent manner. CoPoIFN-a at105units could increase antibody level significantly based on ELISA results, while104and106units of CoPoIFN-a only slightly increased the antibody level. CoPoIFN-a at105units could increase the neutralizing antibody specific to CSFV. These results showed that CoPoIFN-a can increase immunological reaction level induced by pb.5. The crystallization of CoPoIFN-a and computational modeling of CoPoIFN-a/IFNAR2To explore the mechanism for the high biological activity of CoPoIFN-a from structure, we purified CoPoIFN-a from yeast, and concentrated the protein to10mg/mL or20mg/mL. We screened the crystal growth conditions of CoPoIFN-a using commerical crystal screening kits, including Crystal Screen、Crystal Screen2、Index and MembFac.Two protein crystal growth conditions were found. Unfortunately, the diffraction ability of these crystals was poor. We are now trying to refine the conditions to get the crystal with good diffraction quality.Meanwhile, we modeled the ternary structures of CoPoIFN-a or PoIFN-a with its receptors. Due to the high similarity between the terneary structures, we only analyzed the hydrogen bond and hydrophobic interations between CoPoIFN-a and the receptors. In our complex models, we did not observe the direct interactions of the different residues on CoPoIFN-a with the receptor. The original Ser-38, Ser-151and Phe-43in PoIFN-a were changed into Phe, Ala and Leu in CoPoIFN-a, respectively. The side chains of these residues are pointing inside towards the core of the structure, and they changed the local hydrophobicity distribution. Another mutation at position156to arginine seemed more significant, since such replacement introduced more positive potential at the interface in CoPoIFN-a according to surface electrostatic potential analysis. Since introduce of the positive potential on the interface can steer these two proteins to achieve fast or tight binding. On the other hand, it was also possible that mutated residues affected the conformation of the residues nearby that had interactions with the receptor, and consequently enhanced the biological activity of the engineered IFN-a.