热带作物学报2012，Vol.33Issue(12)：2246-2251,6.DOI:10.3969/j.issn.1000-2561.2012.12.024
香蕉束顶病毒Haikou 2分离物DNA2编码框原核表达及抗血清制备
Prokaryotic Expression of DNA2 ORF of BBTV Haikou2 Isolate and Preparation of Polyclonal Antibody
摘要
Abstract
Banana bunchy top disease caused by Banana bunchy top virus which is one of the worst diseases of banana. It is a serious threat to banana production. BBTV genome is composed of at least six circular ssDNA components, and the component DNA2 with less research is the largest variation. About 114 bp of Haikou2 BBTV DNA2 ORF was amplified by regular PCR in this research. The PCR product, digested with restriction enzymes, was inserted into a prokaryotic expression vector pET32a of the same digestion, then recombinant plasmid pET32a-DNA2 ORF was identified and was transferred into E.coli Trasetta(DE3). 12% SDS-PAGE analysis showed that a fusion protein band about 21 ku was detected after induced with IPTG. By the ultrasonic broken instrument, the analysis results showed that the fusion protein was expressed steadily in LB culture and the. optimal expression conditions was 30 ℃, 1.0 mmol/L IPTG with 3 h. The fusion protein was further purified via Ni2+-NTA agarose affinity chromatography, and was used to immune rabbits for antiserum preparation. 0.954 μg/mL purified protein in banana juice was detected and the optimal titer of the antiserum was determined to be 1∶ 25 000 diluted by indirect enzyme -linked immunosorbent assay (ID -ELISA). Western blot analysis- revealed that antiserum was specifically binded the fused protein.关键词
香蕉束顶病毒Haikou2分离物/BBTVDNA2/原核表达/抗血清制备Key words
BBTV Haikou 2 isolate/ BBTV DNA2/ Prokaryotic expression/ Antiserum preparation分类
生物科学引用本文复制引用
谭老喜,王洪星,张雨良,王健华,章绍延,周朋,余乃通,刘志昕..香蕉束顶病毒Haikou 2分离物DNA2编码框原核表达及抗血清制备[J].热带作物学报,2012,33(12):2246-2251,6.基金项目
国家自然科学基金项目(No.31070131) （No.31070131）
国家科技支撑计划课题(No.2007BAD48B01) （No.2007BAD48B01）
中央级公益性科研院所基本科研业务专项(No.ITBB110303). （No.ITBB110303）
