关键词: 香蕉束顶病毒;Rep蛋白;特性分析;纯化体系;建立

Abstract:

With Rep protein from banana bunchy top virus Haikou isolates DNA1 (Accession NO. FJ463042) as the research object, physical and chemical properties, composition, signal peptide, phosphorylation, secondary structure, tertiary structure and functional domains of the protein, etc. were analyzed and forecasted using bioinformatics software, and associated protein purification reagents were configured according to these features. The recombinant plasmid pET32b-Rep of E. coli BL21 (DE3) was inoculated into LB liquid medium and was induced at 20℃, 0.1 mmol/L IPTG for 4 h. A lot of soluble recombinant protein was generated and the supernatant was added to the AKTA Explorer 100 system by affinity chromatography. A different gradient of Washing buffer eluted impurity proteins and products of various stages via 12% SDS-PAGE electrophoresis to determine 100 mmol/L imidazole as washing buffer concentration, and a large number of high-purity recombinant proteins were obtained ultimately. His*Tag Monoclonal Antibody as the first antibody, Western blot showed that the purified recombinant protein was the His-Rep fusion protein. The establishment of this purification system could not only provide basis for studying the crystallization of BBTV Rep, but also provide important data for purification of ABTV, FBNYV, MVDV, PNYDV, etc.

Key words: BBTV(Banana bunchy top virus), Rep protein, characteristics analysis, purification system, establishment