Sequence Analysis Of Five PRRSV Isolates ORF3, 5, 6 Genes And Construction Of The Recombinant Adenovirus Containing ORF3-5 Fusion Gene

Porcine reproductive and respiratory syndrome virus( PRRSV) was one of the most economically important pathogens of swine diease. ORF3, 5, 6 gene of PPPSV GD strains was amplified by RT-PCR and the sequence was analysised in this study. Both the relationship among PRRSV GD isolates each other, and relationship between GD isolates and other strains were discussed. So that to show the basis of origin searching for PRRSV in GD provicine and manufacturing the gene engineering vaccine. The major antigen gene ORF3, 5, 6 of PRRSV were combined to the recombinant adenovirus express system. Based on the extensive infection of adenovirus to body cell of animal, the ORF3-5 fusion gene were developed and iserted into the genome of the adenovirus expressed system inorde to raise the activation of antigen to the system of animal. The paper include two parts: (1)Genetic variation analysis ORF3, 5, 6 genes of PRRSV GD isolates According to the complete sequence of American PRRSV (the ATCC VR2332), three pairs of primers with special restrict enzyme sites were designed. ORF3, 5, 6 genes of PRRSV GD isolates were obtained from 5 PRRSV infected swine samples by RT-PCR, and sequenced after directly cloning into the pMD18-T vector. The sequences of ORF3, 5, 6 genes of GD isolates were compared with the other PRRSV strains in Genebank. The results showed that the sequences of ORF3, 5, 6 of the GD isolates were related closely with ATCC VR-2332 and farther with PRRSV LV strain. And the phylogenetic trees also revealed the GD isolates was related closely with HB-1(sh). (2)Construction of recombinant adenovirus carrying ORF3-5 fusion gene According to the sequence of ATCC VR2332 gene in Genebank, two pairs of PCR primers were designed. The ORF3 gene, a fragment of 780 bp was amplified from the pT-ORF3 by PCR while using WP3L and WP3R, then cloned into pMD 18-T vector and identified by Sal I , Not I/Hind III digestion, and the recombinant plasmids named pMD-ORF3 were obtained. The ORF5 gene were amplified from pT-ORF5 by PCR. The PCR products were cloned into pMD18-ORF3, the recombinant plasmids obtained. The pAdTrack-CMV vector and the interesting gene(ORF3-5)were all digested by the two restriction enzymes Not I and Hind III , then pAdTrack-CMV vector and ORF3-5 gene were linked together with T4 DNA ligase. The ligation mixture was transformed into competent E. Coli DH5a preparaed with Calcium Chloride method. The plasmid DNAs with kanamycin resistant gene were identified by PCR analysis and restriction analysis. The plasmid DNA pAdTrackCMV-3-5 was linearized with Pme I and subsequently cotransfected into the E.Coli BJ5183 together with the backbone plasmid pAdEasy-1. After their homogeneous recombination, screened the positive recombinant adenovirus genome plasmid DNA, the recombinant adenovirus genome DNA was digested with PacI and transfected into 293 cells with Lipofectamine to package the recombinant adenovirus. At last, The recombinant adenovirus carrying ORF3-5 gene was screened under fluorescent microscopy and identified by PCR analysis. The study above offer the scientific basis for the epidemic spread of PRRSV in GD and preventive measures establishing。...