Expression Of Porcine Parvovirus VP2 And NS1 Genes & DNA Immunization With VP2 Gene

Porcine parvovirus (PPV) infection is one of the most important viral diseases, and characterized by reproductive failures in swine. PPV is epidemic in most regions of the world, including China, causing great economic loss in swine industry. Prevention and control of PPV infection is an urgent task.The main non-structural protein of PPV, NS1 encoded by NS1 gene, is associated with the early and late transcription of PPV. NS1 can be used as a differential antigen for differentiating the pigs immunized with "dead" PPV vaccine from pigs infected with wild-type PPV. A PPV designated SY-99 strain was isolated from PK15 cells in 1999. The NS1 gene was amplified from the genomic DNA of SY-99, and then cloned into a prokaryotic expression vector pET28a, resulting in a recombinant plasmid pET28a/SYNS1. The NS1 gene was shown to contain 1989 base pairs (bp) and encode 662 amino acids (aa). A conserved motif GKRN, an important element in PPV transcription, was identified in the deduced NS1 protein sequence of SY-99. There are three potential glycosalation sites in the NS1 protein, at 356-359, 446-449, 513-516aa, respectively. The nucleotide identities of NS1 between SY-99 and PPV strains NADL2-1, NADL2-2 and Kresse are 98%, 99% and 99%, respectively, and the amino acid identities 97%, 99% and 99%, respectively. The pET28a/SYNSl was transformed into E. coli. BL21 (DE3). The NS1 protein was expressed with high-level in the transformed BL21 (DE3) after induction with IPTG. The expressed His-fusion protein was shown to be immunologically active by Western blotting. The IPTG concentration and induction time were optimized. The His-fusion recombinant protein was purified with His'Band column chromatography. The expressed NS1 protein will be beneficial to study of PPV infection.The vaccines available against PPV infection are mostly derived from inactivated virus or modified live virus. These conventional vaccines show many disadvantages, including incidental reversion and less safety and efficacy. So it's essential to develop more effective and safer new-type vaccines against PPV infection.The structural protein VP2 of PPV can form virus-like particles (VLPs). The VLPs are similar in antigenicity to PPV virions, so it can be used as a subunite vaccine candidate for protecting pigs from PPV infection. The VP2 gene of SY-99 was amplified and cloned into a yeast expression vector pPICZaB to obtain a recombinant plasmid pPICZaB/VP2. The pPICZaB/mVP2, modified from pPICZaB/VP2 by site-directed mutation to delete the Sad site in VP2 gene, was linearized by Sad and transformed into the host yeast GS115. The target protein was not detectable by SDS-PAGE analysis after induction by methanol. We mutated some codons of the VP2 gene based on the yeast codon preference. The modified VP2 genewas then cloned into pPICZctA, resulting in pPICZ