| PRRSV is a small enveloped virus with positive-sense single-stranded RNA genome which was easy to emerge variation throughout the genome, especially Nsp2 and ORF5 gene, whose mutation analysis can reflect the whole genome sequence variation.In order to compare the consistency between Nsp2 and GP5 variations ,the Nsp2 and GP5 genes sequence of nine strains PRRSV genome isolated from Henan were analyzed,then exploring the genetic variation characteristics of epidemic PRRSV strains .Besides, porcine IFN-αhas extensive antiviral activity;As porcine IFN-αprotein from eukaryon expressing system gets close to the natural interferon activity,it's of great biological significance in prevention and treatment of PRRSV.Amplifying the Nsp2 and GP5 genes sequences of nine strains PRRSV isolated from different areas of Henan between 2006 and 2009,these genes of NSP2 exhibited different sizes in length ranging from 2850bp to 2937bp and coded 950aa to 979aa, and the length of GP5 were 603bp and coded 200aa. Besides the HeN-3,nucleotide sequences of eight strains PRRSV genome Nsp2 genes were compared to CH-1a,BJ-4,HB-1(sh) HB-2(sh)and VR2332 , the nucleotide sequences homology of NSP2 was 91.1%-92.4%,82.3%-83.1%,94.7%-96% ,87.15-87.9% and 82.6%-83.5%,compared with HN0701and HN0702, the nucleotide sequences homology of NSP2 were 95.45-98.4%,94.1%-95.6%,HeN-3 strains was compared to BJ-4,RespPRRSMLV and VR2332,the Nucleotide sequences homology of NSP2 were 99.2%,99.5% and 99.5% ,Besides the HeN-3,nucleotide sequences of eight strains PRRSV genome GP5 genes were compared to CH-1a,BJ-4,HB-1(sh) HB-2(sh) and VR2332 , the nucleotide sequences homology of GP5 was 95.2%-96.7%,94%-95.2%,95.5%-96.7%,91.9%-92.5% and 88.7%-89.6%,compared with HN0701and HN0702, the nucleotide sequences homology of GP5 were 96.5%-99.2% and 96.7%-99.7%,HeN-3 strains was compared to BJ-4,RespPRRSMLV and VR2332,the Nucleotide sequences homology of GP5 were 98.7%,99% and 99.2%,and the cladogram was drawn up,The results indicated that the genetic variation of Nsp2 and GP5 genes sequences was conformable and nine strains belongs to American type.Through analysising each one of NSP2 and GP5 in the genetic variation, we can obtain information on genetic variation of PRRSV.The lymphocytes were extracted from peripheral blood of healthy porcine,The total RNA were extracted and used to amplificate porcine Interferon-alpha gene by RT-PCR with a pair of primer which was designed according to the genomic sequences of porcine Interferon-alpha in Genebank. The PCR products were connected to PTG19-T vectors,then transformed into DH-5α. The doubtful strains were idenfied by blue-white spot screening, then identified by PCR and enzyme digestion, sequencing by TAKARA. The cloned porcine Interferon-alpha gene contained complete Open reading fram. The results of homology analysis by DNAstar revealed that their nucleotide homology are all over 95.0% .The PTG19-T-pIFNαwere digested by EcoRⅠand XhoI, then IFN-αgene was inserted into eukaryon expression vector pcDNA3.0. The recombinant expression vectors were constructed by PCR and enzyme digestion and sequencing. The recombinant expression plasmid of pcDNA3.0-poIFNαwas transfected into PK-15 cell and selected by G418 resistance. In order to confirm the standard curve and protein expression of porcine IFN-α, the porcine IFN-αELISA kit was used. Establishing the real time PCR method, we obtained mRNA transcription level of porcine IFN-α.,then established stable expression cell lines of porcine IFN-α.In order to identify the antiviral activity ,the normal monocytes cells with porcine IFN-αinterferon were inoculated with PRRV .It revealed that porcine IFN-αpossessed relatively high antiviral activity. |
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