Sequence Analysis Of Porcine Parvovirus SD-68 Strain And Its VP2 Gene Co-expression In Insect Cells With ORF2 Gene Of Porcine Circovirus

NS1 gene of Porcine parvovirus SD-68 strain was cloned and sequenced. Based on the sequence that we got we can conclude that NS1 gene of SD-68 strain is composed of 1989 nucleotides ,encodes a polypiptide of 662 amino acids and shares 99.9%, 99.9%, 99.7%, 98.1% identity of nucleotides and 99.7%,99.5%,99.5%,96.7% identity of amino acids with that of SY-99 strain, Kresse strain, NADL-2(5075) strain and NADL-2(4973) strain, respectively. The distances of NS1 gene of all strains Analyzed by phylogenetic tree, we can conclude that SD-68 strain is near to MVM(i) strain and far to BPV3 strains. The residues of Thr and Ser at the position 435 and 473 in the NS1 gene of SD-68 strain are phosphorylated sites because they are also found at the same position in the NS1 gene of MVM(i) strain.VP2 Gene of Porcine parvovirus (PPV) SD-68 strain was cloned and sequenced .The results showed that VP2 Gene of Porcine parvovirus (PPV) SD-68 strain contained 1740bp and encoded a protein of 579 amino acids .VP2 gene of SD-68 stain exhibited over 99% nucleotide sequence identity and 96% amino acid sequence identity with other PPV strains: Kresse ,SY-99,NADL-2(5075),NADL-2(4973), US-1.Phylogenetic tree analysis of VP2 gene showed that there is the closest phylogenetic relationship between SD-68 strain and kresse strain. The difference between SD-68 strain and kresse strain was the smallest on 378, 383 and 436 residues which decided the tissue tropism of PPV, so the tissue tropism of SD-68 strain was suggested more similar with kresse stain, that is to say, SD-68 was a dermatitis stain. The comparison of amino acid residues of virulent strain (kreese) and vaccine strain (NADL-2 and SD-68) suggested that 215, 378 and 383 were important residues that decided the pathogenicity of PPV.Four DNA sequences of PPV SD-68 strain were amplified by polymerase chain reaction(PCR) with four pairs of primers which were designed according to the complete nucleotide sequence of PPV Kresse strain, NADL-2(5075) strain and NADL-2(4973) strain that were embodied by Genbank. These DNAsequences were cloned and sequenced, respectively. A continuous 4,377 nucleotide sequence spanning structural gene and nonstructural gene was obtained. The comparison of VP1, VP2, NSland NS2 gene of SD-68 strain with PPV Kresse strain, NADL-2(5075) strain and NADL-2(4973) strain indicated that VP1 gene of SD-68 strain shared 99.4%, 99.4%, 98.4% homology with the three strains, respectively;, identity of VP2 gene of SD-68 strain with them were 99.3%, 99.2%, 99.0%, respectively; identity of NS1 gene of SD-68 strain with them were 99.9%, 99.7%, 98.1%, relatively, identity of NS2 gene of SD-68 strain with them were 99.8%, 99.4%, 93.8%, relatively. The Phylogenetic analyses showed the relativeness of SD-68 strain and Kresse strain was closest, So SD-68 strain was probably a mutation that the virulence of kresse strain was weakened because of mutations of residues of VP2 that decided the virulence of strains.A recombinant baculovirus expressing Porcine parvovirus (PPV) vp2 gene and Porcine circovirus (PCV) orf2 gene was constructed with Bac-to-Bac Baculovirus Expression Systems. After transfecting the recombinant virus into Sf9 cells for 3 days, PPV vp2 and PCV orï¿¡2 can be detected by indirect immunofluorescence antibody assay (IFA) and Western blot. The results show that the viral protein 2(vp2) gene of PPV and orf2 gene of PCV were successfully expressed in insect cells.