Prokaryotic Expression, Preparation of Polyclonal Antibody and Immunodetection of HearNPV orf86

Abstract:

HearNPV orf86 is a function unknown gene of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). The coding region of orf86 was amplified from the HearNPV genome by PCR and then was inserted to the prokaryotic expression vector pGEX-4T-2. The recombinant plasmid was transformed into E. coli BL21 to express the GST fusion protein in the bacteria. The purified fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody. The titer of the anti-HearNPV ORF86 polyclonal antibodies reached to 1 : 5.12 × 105 determined by ELISA. A 36 kD protein was identified by Western blot on HZAM1 cells infected by HearNPV using the prepared polyclonal antibody, which the protein size was consistent with the predicted protein size encoded by orf86. The results indicated that the polyclonal antibodies against ORF86 was successful prepared and it would facilitate the detection and related study on ORF86.

Key words:Helicoverpa armigera nucleopolyhedrovirus (HearNPV), ORF86, Prokaryotic expression, Polyclonal antibody