Cloning And Prokaryotic Expression Of Nonstructural Protein NS2 Gene Of Goose Parvovirus

Goose Provirus (GPV) is the pathogen of highly contagious and fatal infectious diseases of goslings and Muscovy ducklings. The typical pathological lesions were acute intestine inflammation. Generally it was called goose plaque or goose parvovirus infectious. The pathogen was first isolated by Fang ding-yi in China in 1961. Up to date. GP is influencing in China, it make large harness for feeding-geese.GPY has been classified as a member of the parvovirus genus of the parvoviridae. No helper virus is required for replication of GPV. The genome of goose parvovirus is a 5106nt linear single-stranded DNA, it contains two open reading frames (ORF) and is flanked by inverted terminal repeats (ITRs). The right ORF encodes three structured proteins (VP1, VP2, VP3), and the left ORF encodes at least two nonstructural proteins that are involved in regulating all aspects of the viral life style. But the comparison amino acid sequence shows that GPV DNA has high homology with the human adeno-associated virus AAV-2 in dependovirus and B19 in erythrovirus. The close genetic relationship of goose parvoviruse and AAV allows the examination of the molecular biological properties of the nonstructural proteins of GPV. After the GPV infected the cell the viral life cycle was regulated by the nonstructural proteins encoded by the virus.According to the published of GPV B strain genome nucleotide sequences in genbank and a pair of specific primers were disigned with Oligo4.1. Full-length NS2 gene was amplified by polymerase chain reaction (PCR). The interest gene was cloned into pMD18-T vector and sequenced. It reveals that NS2 gene contains 1356 bases which encodes 451 amino acid which is the same as GPV B. Comparing with GPV B strain there are 17 nucleotides and 6 amino acids variation. Among the 17 different nucleotides only 6 nucleotides induce amino acids mutation. Sequence analysis shows that they share 98.75% similarity at the DNA, and 98.67% at the protein level.NS2 gene was cloned into the multiple cloning sites of prokaryotic expression vector pGEX-6P-l. The recombinant plasmid PGEX-6P-NS2 was constructed and transformed to the competent cell BL21 (DE3) plysS, Positive bacterium strain was induced by IPTG. SDS-PAGE reveals that the NS2 gene was expressed in E.coli BL21 (DE3) plysS with high efficiency. The weight of product is 75ku, which is fussed with GST. The expressed protein accounts for 30% of the total bacterial protein.This study provided the basis for the further research about GPV and the physical, chemical character and biological activity of NS2. It was prepared for the development of GPV diagnostic reagent. And it also provided the evidence for taxonomy of GPV and its relationship in evolution.