Cloning of BBTV-HN DNA2 ORF and Preparation of Polyclonal Antibody
The Opening Reading Frame fragment of Hainan isolate(BBTV-HN) DNA2 of banana bunchy top virus in Hainan Island was cloned by PCR and fused to the downstream of gulutathion-S-transferase gene in the expression vector pGEX-6p-1. After the correct encoding region was confirmed by sequencing,the recombinant plasmid pGEX-HN-B2 was induced by isopropyl-B-D-thiogalaotopyranoside (IPTG) in E.coli Rosetta (DE3). Compared to the control without inducing,a new protein band of 33 Ku was found in the induced products after running the SDS-PAGE. To prepare antiserum against the fused protein,the 33 Ku protein was used to immunize rabbit. Western blot analysis revealed that the antiserum against DNA2 encoding protein was obtained. This will help us understand the role of DNA2 encoding protein in the infected tissues.