香蕉条斑病毒ORFⅠ、ORFⅡ在大肠杆菌中的原核表达和自激活特性的鉴定  

The Prokaryotic Expression and the Test of Self-activation of ORFⅠand ORFⅡ of Banana Streak Virus in E.coli

作　　者：林湛松[1,2] 王健华[1] 张雨良[1] 江林[1,2] 杨文君[1] 余乃通[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,海口571101 [2]海南大学环境与植物保护学院,儋州570228

出　　处：《基因组学与应用生物学》2010年第6期1072-1077,共6页Genomics and Applied Biology

基　　金：国家自然基金项目(31070131);中央级公益性科研院所基本科研业务费专项(ITBBZD0754)共同资助

摘　　要：目前,香蕉条斑病毒所有3个ORF表达产物功能均不明确,通过双杂技术研究病毒未知蛋白与宿主蛋白的互作,可以初步推断未知蛋白的功能。本实验旨在构建香蕉条斑病毒ORFⅠ和ORFⅡ在细菌双杂系统中的诱饵质粒。首先以课题组保存的连接有香蕉条斑病毒河口分离物全基因组的pMD-18T载体DNA为模板,经过PCR扩增,切胶回收,并通过与带有λcI基因的pBT载体共同双酶切及T4连接酶连接后,得到与λcI基因融合表达的重组载体pBT-ORF1和pBT-ORF2。转化大肠杆菌XL1-BlueMRF'报告菌株,用IPTG(0.1mmol/L)诱导目的基因片段表达。Westernblotting结果表明,ORF1-λcI和ORF1-λcI两种融合蛋白均成功表达,且大小与预期一致。之后,pBT-ORF1及pBT-ORF2分别与空质粒pTRG共转化XL1-BlueMRF'报告菌株,pBT空质粒和pTRG-Gal11p共转化作为阴性对照。结果显示pBT-ORF1及pBT-ORF2均无自激活现象,可以进行后续的细菌双杂工作。本实验为BSV与宿主的细菌双杂系统的建立及蛋白组学的研究奠定了基础。The function of all the proteins expressed by the three ORFs of Banana streak virus have lack of research,and we can get a rough idea on it by means of two-hybrid system.In this study,we are aimed to construct the bait plasmids of ORF Ⅰand ORF Ⅱ utilized in the bacterial two-hybrid system.The two target segments were firstly generated by PCR amplification,using the whole genome of BSV-Yunnan as the template.Then after double digestion and ligation by T4 ligase with pBT plasmid which carries the λcI gene,the recombinant plasmid pBT-ORF1 and pBT-ORF2 were generated.The plasmid was thereafter transformed into the competent cell of E.coli XL1-Blue MRF1＇,and the E.coli cells were then induced by IPTG at the concentration of 0.1 mmol/L to express the fusion protein.Western blotting showed that,the two fusion proteins could be expressed by E.coli XL1-Blue MRF1＇,and both of them had right molecular weight.Then both of the recombinant plasmids were co-transformed into E.coli XL1-Blue MRF1＇ with empty plasmid pTRG respectively,meanwhile,the pBT empty plasmid and pTRG-Gal11p were also co-transformed into E.coli XL1-Blue MRF1＇ as the control sample.The results suggested that both pBT-ORF1 and pBT-ORF2 could not self-activate in E.coli,and thus could be utilized in the screening of bacterial two-hybridization by BacterioMatchⅡ Two-Hybrid System.

关 键 词：香蕉条斑病毒 原核表达 细菌双杂交 WESTERN BLOTTING 自激活鉴定 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治] Q939.4[农业科学—植物保护]