Research On Characteristics Of Recombinant Porcine OAS1Anti-Japanese Encephalitis Virus

2’-5’oligoadenylate synthetases (OAS) is an important component of innate immune system of mammal, and the2’-5’-oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. Its essence is a kind of antiviral protein induced by interferon. Intracellular OAS/RNaseL signaling pathway plays an important role in host innate immune response by controlling flavivirus, reovirus and encephalomyocarditis virus. In addition, in recent years, studies have confirmed that RNaseL-independent pathway is exist during antiviral effect of the OAS family. The OAS protein can directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. OAS genes are found to be biological marker genes for resistant to flavivirus infection among mice, horses and human beings, but the molecular mechanism has not yet be en clarified. However, the report about porcine OAS gene research is less. The amino acid homology between porcine OAS1protein and anthropogenic OAS1protein was73%, and their enzymology characteristics were similar. Therefore, further research about both whether there are biological marker genes which are resistant to flavivirus (JEV) infection in pigs or not and whether it is OAS which is the risk marker gene of disease caused by JEV infection in pigs or not are necessary. In view of serious damage caused by Japanese encephalitis virus(JEV) to China and its wide transmission range in China; The JEV disease have lead to serious economic loss in the pig industry, and also because pigs play an important role in JEV propagation as an intermediate host, the research about the role of porcine OAS played in resistance to JEV infection and its molecular mechanism are of great significance. The design of this study mainly includes the following content:1. Research about the expression of soluble recombinant porcine OAS1protein and its physical and chemical properties Based on a published porcine OAS1gene sequence, we design primers and use the method of RT-PCR to amplify porcine OAS1gene from PK cells, its length is1050bp. Purified gene sequence is ligated into the pMD18-T Easy vector and identify its correction through cloning sequencing. The objective gene of correct sequence was connected with pET28a (＋) vector and then we can construct prokaryotic expressed recombinant plasmid, convert it to e.coli Rosetta2(DE3). Induce it by IPTG at O.lmmol/L and lower temperature for a night, the results of SDS-PAGE showed that we obtained a large amount of porcine OAS1proteins with good solubility. The molecular weight of protein is about40KD. Through His labels on the vector, we used nickel column to purify expressed protein with the theory of affinity chromatography. The result is that a large amount of stable and relatively pure protein was obtained. The identification results of SDS-PAGE showed that the purity of protein is above95%.Use the purified objective protein to immune mice for the preparation of specificity serum, the result of western blot showed that there is an apparent specificity strip after the interaction reaction of antiserum with porcine OAS1protein. This study has laid a foundation for the effect appraisal and the mechanism research of antiviral effect of porcine OAS1protein.2. Analysis of the study about antiviral properties of recombinant porcine OAS1proteinPurify porcine OAS1protein from prokaryotic expression, using desalination column to deal with the protein. The result of MTT colorimetry test showed that the OAS1protein which have dealed by the desalting treatment had little effect on cell activity in the condition of low concentration. Detect the activity of porcine OAS1protein in inhibiting virus infecting cells by Real Time PCR method. The important thing is to detect the antiviral effect of porcine OAS1protein anti-pseudo rabies virus (PRV) in PK-15cell lines,the antiviral effect of porcine OAS1protein anti-porcine respiratory and reproductive syndrome virus (PRRSV)in Marc-145cell lines, and the antiviral effect of porcine OAS1protein anti-Japanese encephalitis virus (JEV) in BHK21cell lines by Real Time PCR method. Results showed that porcine OAS1protein from exogenous expression has a wide range of antiviral activity; they both have the activity of inhibiting virus infecting cells in DNA and RNA viruses. The cells which were pretreatment with protein can significantly reduce the levels of viral DNA or mRNA in the cell. And the effect of inhibition of virus infected cells is a dose-response relationship. In terms of our test results, especially the protein inhibited JEV infection in BHK-21cells on the most significant effect. When the concentration of protein is200μg/mL, mRNA level of JEV in cells of control group is8.04times the level of mRNA of JEV in cells of protein-pretreatment group. Use porcine OAS1protein to deal with cells at various stages during the virus infecting cells, to detect the best action time of porcine OAS1proteins inhibiting virus infecting cells by using the method of Real Time PCR and plaque reduction. Results show that cells which are pretreatment with protein with the concentration in100μg/mL can significantly inhibit cells from infected by virus, and had an effect on replication of virus in cells, which reduce the number of progeny virus. Protein had little impact on virus itself, cells which processed by protein can hardly inhibit the virus infection, and have no obvious therapeutic effect on cells infected by virus. With protein processing infected cells after poisoned, we found that the porcine OAS1protein (lOOμg/mL) still has certain activity of inhibiting virus infecting cells, but the effect was not obvious. Therefore, porcine OAS1protein mainly play its role in inhibiting virus infecting cells in the way of using it for cells pretreatment, for the activation of downstream signaling pathways, prevented proliferation of virus in cells, proteins have little impact on virus itself, and have no obvious antiviral effect on infected cells. This research has laid a foundation for our further study on the antiviral mechanism of porcine OAS protein in vitro.3. The study about mechanism of effect of recombinant porcine OASl anti-japanese encephalitis virusAccording to analysis about the antiviral spectrum and the best time of antiviral effect of porcine OAS1protein in vitro, which expressed extracellularly, we have carried on a further discussion about its mechanism of anti-japanese encephalitis virus. Using the cell which was pretreatment with porcine OAS1protein to detect the effect of antiviral infection of porcine OAS1protein in cells by controlling the inoculation quantity of virus and the effect time after cell inoculated with virus, and we appraise the effect at the protein level, genome level and viral titers, respectively. The results showed that porcine OAS1protein can effectively play its role in the effect of antiviral, mainly through the inhibition of viral replication in cells. But even at higher protein concentration (200μg/mL), porcine OAS1protein had no effect to antiviral at the period of virus and cell interaction, and porcine OAS1protein almost have no effect in the absonption of virus to cells, they could not control the quantity of adsorptive virus to cells effectively, and even can not prevent virus invading into the cells. Using porcine OAS1protein to pretreat the cell, viral mRNA and viral titer were slightly decreased after6h of inoculated virus. But the viral titer of the supernatant of infected cell had significantly decreased after12h of inoculated virus, and the intracellular viral mRNA also had significantly decreased. Compared to the positive control, viral protein content was significantly decreased in protein treatment group. Porcine OAS1protein could significantly inhibit the replication of virus in cells after24h of inoculated virus. In comparison with the positive control group, whether the viral titer of the supernatant of infected cell or the intracellular viral mRNA had significantly decreased. Protein treatment group virus protein content compared to the positive control also have significantly decreased, and the viral protein content of protein treatment group was significantly decreased to compared with the positive control, and when the protein concentration at200ng/mL, we still could not detect the viral E proteins. The mainly dependent amino acid residues of OAS/RNaseL pathway are the D74and D76amino acid residues of porcine OAS1protein, which determine the catalytic activity of RNaseL. This study produce mutant of porcine OAS1protein D74and D76amino acid residues by using technology of point mutations by the Quick change in the PCR, which have laid a good foundation of the study about if there are multiple molecular signaling pathways of the porcineOAS1protein. Results showed that the mutant of porcine OAS1still had antiviral activity, and In comparison with its parent porcine OAS1protein, the mutant was slightly decreased in the effectiveness of antiviral infection, and it is also dose dependent. It seems that porcine OAS1proteins effectively play its role in the effect of antiviral, mainly through the inhibition multiple molecular signaling pathways.