摘要
为揭示水稻瘤矮病毒(RGDV)外层衣壳蛋白P8的结构特点及其在大肠杆菌中的表达特性。利用生物学软件和在线分析工具,对RGDVP8蛋白结构特征进行了生物信息学分析;同时将通过RT-PCR扩增的P8基因克隆到pGEX-4T-1上,构建pGP8转化大肠杆菌E.coliBL21(DE3),在IPTG诱导下表达。利用表达的融合蛋白免疫家兔,制备了抗血清,并以RGDV、水稻矮缩病毒(RDV)、水稻条纹病毒(RSV)、水稻锯齿叶矮缩病毒(RRSV)4种病毒作为供试材料进行特异性检测。分析显示P8蛋白在C端位置有一个典型的跨膜螺旋区,整个蛋白也具有Phytoreo P8家族共有的典型结构域Phytoreo P8 domain。SDS-PAGE和蛋白印记分析表明,RGDVP8融合蛋白分子量约为73kDa,以包涵体的形式获得了高效表达。获得的效价高、特异性强的抗血清,为水稻瘤矮病毒的快速检测及P8蛋白的功能研究奠定了基础。
To elucidate structural characters and expression characters of RGDV P8 protein in E.coli, bioinformatics analysis were performed by using biology software and analysis tools online. P8 gene was successfully amplified by RT-PCR and inserted to pGEX-4T-1 expression vector. The protein was then expressed in E. coli BI21 （DE3） with IPTG induction. The antiserum was produced by immunizing rabbit with fusion protein, and the specificity of antiserum was evaluated by detecting RGDV, RDV, RSV, RRSV. Analysis showed that P8 protein had a typical transmembrane helices region, and a Phytoreo P8 domain shared with Phytoreo P8 family. SDS-PAGE and Western blotting analysis showed that molecular weight of P8 fusion protein was about 73 kDa. P8 protein was efficiently expressed in the form of inclusion body. The antiserum with high titer and specificity provides the basis for detecting RGDV and studying function of P8 protein.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2009年第8期51-56,共6页
China Biotechnology
基金
2007年公益性行业(农业)科研专项(nyzx07-051)
福建省自然科学基金计划(2006J0065)资助项目
关键词
水稻瘤矮病毒
P8蛋白结构分析
融合蛋白
抗血清
Rice gall dwarf virus P8 protein Structure analysis Fusion Protein Antiserum
