香蕉条斑病毒云南分离物ORFⅠ的原核表达及其抗血清制备  

Prokaryotic Expression of ORFⅠof Banana Streak Virus Yunnan Isolate and Preparation of a Virus-specific Antiserum

作　　者：王健华[1,2] 林湛松[1,2] 张雨良[2] 杨文君[1,2] 余乃通[1,2] 刘志昕[2] 

机构地区：[1]海南大学环境与植物保护学院,海南儋州571737 [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技术重点开放实验室,海南海口571101

出　　处：《热带作物学报》2011年第7期1356-1359,共4页Chinese Journal of Tropical Crops

基　　金：海南大学“211工程”建设项目;中央级公益性科研院所基本科研业务费(No.ITBB110304)

摘　　要：以连接在pMD-18T载体上的香蕉条斑病毒云南分离物(BSV)全基因组为模板,设计特异引物,PCR扩增获得ORFⅠ全长序列。目的片段及原核表达载体pET32(b)分别经EcoRⅠ和XhoⅠ双酶切后,连接构建重组质粒pET32-ORFⅠ,将鉴定为正确的重组质粒转化E.coli BL21(DE3)感受态细胞,获得原核表达OFRⅠ工程菌。工程菌在28℃条件下,用浓度为0.1 mmol/L的IPTG诱导表达6 h,离心收集菌液并超声波破碎,获得以含目的肽段的可溶性蛋白为主的融合蛋白。将融合蛋白通过Ni2+-NTA亲和层析柱纯化后,得到纯度较高的融合蛋白。以此蛋白为抗原免疫家兔,获得特异抗血清,该抗血清的效价达1∶100 000,Western Blot验证表明,抗血清的特异性较强。本研究为BSV的快速检测以及BSV编码蛋白的功能研究奠定了基础。The ORFⅠsegment was amplified by PCR utilizing the whole genome of BSV Yunnan isolate as the template,then subcloned in the prokaryotic expressional vector pET-32b（＋）simultaneously digested by both EcoRⅠand XhoⅠ.The recombinant plasmid pET32-ORFⅠ was then transformed into the competent cells of E.coli BL21（DE3）.Then the genetic engineering bacterial strain harboring pET32-ORFⅠ was induced by 0.1 mmol/L IPTG at 28 ℃,and the soluble fusion protein including the ORFⅠprotein was generated and then purified by the Ni2＋-NTA column.The purified protein was then utilized to immune a rabbit in order to obtain the virus-specific antiserium.The tilter of the antiserium by indirect enzyme-linked immunosorbent assay（ID-ELISA）was up to 1∶100,000.The antiserium was proved to have high specificity by Western Blot.The present study had paved a way to establish the methods of BSV detection and the research protein function of BSV.

关 键 词：香蕉条斑病毒 原核表达 抗血清 

分 类 号：S668.1[农业科学—果树学]