香蕉束顶病毒海口分离物CP基因的原核表达、纯化及其抗血清制备  

Prokaryotic expression and Purification of CP gene of Banana Bunchy Top Virus HaiKou Isolate and Preparation of Antiserum Against its CP

作　　者：余乃通[1,2] 冯团诚[1] 王健华[1] 张雨良[1] 刘志昕[1] 

机构地区：[1]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技术重点开放实验室,海南海口571101 [2]海南大学环境与植物保护学院,海南海口570228

出　　处：《热带作物学报》2010年第5期767-771,共5页Chinese Journal of Tropical Crops

基　　金：国家科技支撑计划项目(No.2007BAD48B01);教育部/海南省教育厅联合资助博士点基金项目(No.20050565002)

摘　　要：用PCR方法从感染香蕉束顶病毒(Banana bunchy top virus,BBTV)的香蕉植株幼嫩假茎和叶片总DNA中扩增外壳蛋白(coat protein,CP)基因。回收目的片段,经XhoⅠ和BamHⅠ双酶切后与同样酶切的质粒载体pET30a(+)相连接,酶切验证及测序结果获得了含BBTV CP基因的重组质粒pET30a-CP。将重组质粒转化Codon plus表达菌,在25℃、0.4mmol/L IPTG条件下诱导6h或过夜,经12%SDS-PAGE电泳分析可知,该重组菌表达了一个与预期分子量大小一致的His-CP融合蛋白,约为30ku,且主要以包涵体的形式存在。多次洗涤包涵体后得到高纯度的融合蛋白,用其免疫家兔,获得BBTV CP蛋白抗血清。间接ELISA法测定抗血清效价大于51200。Western blot分析表明,抗血清能与融合蛋白特异性结合。The coat proteins gene （CP） was amplified from total DNA isolated from young pseudostems and leaves of banana （Musa paradisiaca） plants infected with Banana Bunchy Top virus by polymerase chain reaction （PCR）.The CP gene was digested by Xho Ⅰ and BamH Ⅰ , and inserted into the prokaryotic expression vector pET-30a （＋）.The recombinant plasmid pET30a-CP was identified by enzymatic digestion and gene sequencing.Escherichia coli codon plus containing the recombinant plasmid was induced at 25 ℃ with 0.4 mmol/L IPTG for 6 h or over night.And the result was that an expected 30 ku His-CP fusion protien was expressed as inclusion body analyzed with 12% SDS-PAGE electrophoresis.Inclusion body was washed several times and obtained high purity of fused protein.The purified recombinant protein was used to immunize rabbits for production of antiserum against BBTV CP.The titer of the antiserum was higher than 1 ：51 200 determined with indirect enzyme-linked immunosorbent assay（ID-ELISA）.Western blot analysis revealed that antiserum specifically binded the fused protein..

关 键 词：香蕉束顶病毒 CP基因 原核表达 纯化 抗血清 

分 类 号：S463[农业科学—植物保护]