Cloning Of BBTV DNA2 ORF And Preparation Of Polyclonal Antibody

Banana is one of the five important fruit in the world.It plays an very important role in the international trades.Banana bunchy top disease(BBTD) is one of serious diseases of banana which intected by banana bunchy top virus(BBTV).Molecular biology research of BBTD began more than 20 years ago.The virus has small isometric virions of 18-20nm.The genome consists of at least six components of circular ssDNA about 1.0～1.1kb.A further study about the compents of BBTV in the world,though,whether ORF of BBTV DNA2 encoding protein and the function in the whole plant.is unknown.In order to express he Opening Reading Frame fragment of Banana bunchy top virus Hainan isolate (BBTV-HN)DNA2 correctly ,a pair of primers were designed in accordance with the reported BBTV-HN DNA2 gene sequence.The Opening Reading Frame fragment of BBTV-HN DNA2 was cloned by PCR using total DNA from banana tissues which are of characteristic of typical BBTV symptom as template.and fused to the downstream of gulutathion-S-transferase gene in the expression vector pGEX-6p-1.After confirming the correct encoding region by sequencingthe recombinant plasmid pGEX-6p -B2 was induced by isopropyl-B-D-thiogalaotopyranoside(IPTG) in E.coli Rosetta(DE3). Compared to noninducing control, a new protein band of 33ku was found in the induced products after running SDS-PAGE. For getting antiserum against the fused protein, the 33KDa protein was used to immunize rabbit. Western blot analysis revealed that the antiserum against DNA2 encoding protein was obtained. This will help us to understand the role of DNA2 encoding protein in the infected tissues.