| Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases affecting the swine industry worldwide, but its affection and pathogenic mechanism remain to be study. The PRRSV genome codes for seven structural proteins and 14 nonstructural proteins. Among the nsps, Nsp4 contains serine protease, accounting for cleaving Nsp3-Nspl2. Nsp4 plays an important role in PRRSV affection and replication. In this study, purified recombined Nsp4 and Nspl2 proteins were usd to immunize BALB/c mice, and polyclonal antiserum were obtained successfully. pEGFP-Nsps and pcDNA-BB0907/NT0801/SlNsp4 were constructed to screened for IFN-β suppression. In addition, the transcription factor IRF3 was cloned into pcDNA3.1 to stimulate pIFN-P promoter. The effects on IRF3 stimulated-pIFN-β promoter activity by Nsp4 from different strains were also analysed in current study. The details as follow:1. Preparation and identification of antibody to Nsp4 and Nspl2 of PRRSVIn this study, PRRSV Nsp(nonstructural protein)4 and Nspl2 genes were cloned into pET32a(+) and pGEX-6P-1, respectivly. The recombinant plasmids were expressed in E.coli BL21(DE3) after induction by IPTG. The recombinant Nsp4 protein was mainly soluble with molecular weight of 42kD, and the recombinant Nsp12 protein was mainly in inclusion form with molecular weight of 44kD. Western-blot showed that both of them had highly reactogenicity. The recombinant Nsp4 and Nsp12 protein were purified through Ni-chelating affinity chromatography and inclusion body lotion, after which they were used to immunize BALB/c mice, respectively. The obtained antiserum could specifically react with the antigen of PRRSV in Western-blot and IFA, and they laid a foundation for further functinonal research for Nsp4 and Nsp12 proteins.2.The effect on pIFN-β promoter activity by PRRSV Nsp4Four Nsps have been identified to contain the type IIFN suppressive activities:Nsp1,Nsp2, Nsp4, and Nspl 1 lately. Considerable progress has been made for mechanism of type IIFN suppressive effect by Nsp1,2 and 11 other than Nsp4. In the study, ten Nsp genes of PRRSV NT0801 were cloned into pEGFP-N1, and then transient transfected into BHK-21 and Hela cells. The results from fluorescence microscope and Western blot showed that all the recombinant plasmids could be expressed in BHK-21 and Hela cells. After that, Dual Luciferase Reporter were used to study the effect on poly(I:C)-stimulated pIFN-P promoter activity by pEGFP-Nsps in Hela cells. The results showed that Nspl, Nsp2 and Nspll had obvious inhibition on promoter activity (P< 0.001), but Nsp4 had hardly any effect on pIFN-p promoter activity. Nsp4 from strains with different virulence(BB0907,NT0801,Sl) were cloned into pcDNA3.1, and then identified by Western Blot. Results from Dual Luciferase Reporter showed that pcDNA-BB/NT/SlNsp4 expressed in Hela/Marc-145 cells had no effect on pIFN-P promoter activity stimulated by either poly(I:C) or IRF3. The results showed that PRRSV Nsp4 had no immunosuppressive effect, which was different from other study, making an important contribution to the study of PRRSV immunosuppression. |
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