鱼类淋巴囊肿病毒32kDa黏附蛋白的特性分析.pdfVIP

The expression plasmids of pET-32a-ORF038 was constructed and transformed into Escherichia coli strain BL-21(DE3), and recombinant protein with molecular weight 50kDa was obtained by induction of IPTG and purified by Ni-NTA column. Polyclonal antibodies against the recombinant protein were prepared by immunization of rabbits. ELISA results showed that polyclonal antibodies could specifically bind to the recombinant protein; In Western blotting, the antiserum could strongly react with the recombinant protein as well as a 32kDa protein in LCDV, which indicate that the antiserum possessed high specificity. Colloidal gold marked polyclonal antibody against ORF038 recombinant protein was used as a probe to locate the 32kDa protein encoded by ORF038 in LCDV. The Immunoelectron microscopy results showed that gold particles were located at the outermost surface of LCDV particles, indicating that the 32kDa protein was the envelope protein of LCDV.In an immunofluorescence assay, the recombinant protein tagged with FITC was incubated with FG cells and positive signals were detected primarily on the cell membrane, indicating that the recombinant protein could bind to the membrane protein of FG cells. Blocking ELISA results showed that the blocking rate of MAb against 27.8kDa receptor protein was 63.2%; In a neutralization assay, pre-incubation of LCDV with the polyclonal antibodies against the recombinant protein could largely impair the virulence of LCDV and inhibit LCDV infection to FG cells in vitro, showing a dose-dependent blocking effect, which further indicate that the 32kDa protein was the vir