Expression,Purification,Preparation of Polyclonal Antibody and Subcelluar Localization of the NS2 Protein of Periplaneta fuliginosa Densovirus / 微生物学通报

ABSTRACT

NS2 is a nonstructural protein of Periplaneta fuliginosa densovirus(PfDNV) with a molecular mass of 30 kD,whose function is not yet clearly understood. In order to study the expression,subcellular distribution and the function of NS2 protein,the coding region of NS2 was amplified from the hindgut tissue of cockroaches infected with PfDNV by RT-PCR and then the recombinant prokaryotic expression vector pET28a-NS2 was constructed. The recombinant plasmid was transformed into E. coli BL21(DE3) to express the 6?His fusion protein in the bacteria. After purification,the fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody. The specificity of the anti-NS2 antibody was successfullyproved by western blotting on the eukaryotic expressed products of NS2 protein.Meanwhile,the full sequence of ns2 gene was also cloned into the eukaryotic expression vector pAC. The recombinant plasmid pAC-NS2 was then transfected into Schneider line 2(S2) cells to express NS2 protein in the insect cells. The subcellular localization of NS2 in the insect cells was then investigated by indirect immunofluorescence technique using the anti-NS2 polyclonal antiserum. The confocal laser scanning microscope observation showed that NS2 protein was located primarily in the cytoplasm with some punctate nuclear staining.