香蕉束顶病毒Haikou2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

作　　者：谭老喜[1,2] 王洪星[2] 张雨良[2] 王健华[2] 章绍延[1,2] 周朋[2] 余乃通[2] 刘志昕[2] 

机构地区：[1]海南大学环境与植物保护学院,海南海口570228 [2]中国热带农业科学院热带生物技术研究所/农业部热带作物生物学与遗传资源利用重点实验室,海南海口571101

出　　处：《热带作物学报》2012年第12期2246-2251,共6页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31070131);国家科技支撑计划课题(No.2007BAD48B01);中央级公益性科研院所基本科研业务专项(No.ITBB110303)

摘　　要：香蕉束顶病毒(Banana bunchy top virus,BBTV)是危害香蕉的主要病毒之一,严重威胁着香蕉的生产。BBTV基因组至少由6个环状ssDNA组分组成,其中DNA2组分变异最大,是否编码蛋白及其功能缺乏研究。本研究通过PCR方法克隆了BBTV Haikou2 DNA2 ORF,将其构建到pET32a载体中,进行原核表达和抗血清制备。结果表明重组表达载体pET32a-DNA2ORF在表达菌Transetta(DE3)中表达一个约21 ku的特异融合蛋白,通过超声波破碎仪破碎细胞和诱导条件优化,分析表明,该蛋白主要以可溶形式存在,最佳表达条件为30℃、1.0 mmol/L IPTG诱导3 h。融合蛋白经过Ni2+-NTA琼脂糖亲和层析纯化,免疫家兔制备出抗血清。Western blot实验表明抗血清具有特异性;酶联法(ID-ELISA)测定表明,抗血清效价达到25 000,能够检测出香蕉汁液中加入的0.954μg/mL浓度的表达纯化蛋白。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 DNA20RF 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-DNA20RF was identified and was transferred into E.coli Trasetta（DE3）. 12% SDS-PAGE analysis showed that a fusion protein barld 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 Ni^2＋-NTA agarose affinity chromatography, and was used to immune rabbits for antiserum preparation. 0.954 p,g/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 

分 类 号：Q78[生物学—分子生物学] S432.41[农业科学—植物病理学]