Molecular Characterization Of Two Porcine Parvoviruses And Prokaryotic Expression Of The Main Proteins

Porcine parvovirus, prevailing in the swine herd, could lead to the reproductive failure of sows andcause economic losses to the swine industry in the world. At present, the studies about PPV mainlyfocused on the structural protein VP2 and non-structural protein NS1, but little on the whole genome.There were only two complete genome sequences recorded in GenBank (accession no. U44978 andNC₀01718). For this purpose, two PPV isolates (LXB-PPV-20-06 and LXB-PPV-16-06) weresequenced to analyze the molecular characterizations of the whole genome and have an analysis on thegenetic variation of PPV compared with the sequences recorded in GenBank. Furthermore, the nonstructuralprotein NS1 and structural protein VP2 were expressed by E. coli.Seven pairs of primers which cover the whole genome were designed to amplify the whole genomefragments by PCR reaction. The positive recombinant plasmids were sent to TaKaRa Biotechnology(Dalian) CO., LTD. for sequencing. The results show that the lengths of LXB-PPV-20-06 and LXB-PPV-16-06 are 5013nt and 5031nt, respectively. There is no insertion or deletion in the coding region.However, some deletions exist in the non-coding region nearby the VP1/VP2 stop codon, which lack 63and 45 nucleotides, respectively. This may be relevant with the virulence. The sequence alignments ofNS1 gene and coding protein show that LXB-PPV-20-06 strain has high homology with AmericanKresse strain and Chinese NJ strain. PPV-16-06 strain has high homology with Chinese SY-99 strain,which has only one amino acid difference. The sequence alignments of VP2 gene and coding proteinshow that LXB-PPV-20-06 strain has high homology with American Kresse strain, whose codingsequences are the same with the Kresse strain. LXB-PPV-16-06 strain has high homology with LJL-12strain. According to the sequence alignment results and the variations of amino acids in position 215,378, 383, 436 and 565, the clinical pathological type of PPV infection is relevant with these differences.The genetic variation analysis shows that the PPV strains prevailing in our country adjoined each other.The NS1 gene and VP2 gene of LXB-PPV-20-06 strain were amplified by PCR, digested byrestriction endonucleases, and cloned to pET-32a (+) vector. The recombinant plasmids were identifiedby PCR, double digestion and sequencing. The positive recombinant plasmids pET-NS1 and pET-VP2were screened and then transformed into E. coli Rosetta (DE3). The recombinant bacteria were inducedby 1mmol/L IPTG. The expressed products were analyzed by SDS-PAGE. The results show that the NS1protein and VP2 protein were both expressed in E. coli. The recombinant proteins were purified by Ni-NTA resin. The molecular weights of NS1 protein and VP2 protein are 97kDa and 82 kDa, respectively.The western blot results show that the target proteins have good antigenicities, which lay the foundationfor the application of NS1 and VP2 protein in the clinical diagnosis.