水稻条纹病毒CP与叶绿体Rubisco SSU引导肽融合基因的构建及其原核表达  

Construction of Fusion Gene of Rice stripe virus (RSV) CP and Rubisco SSU Leader Peptide of Rice Chloroplast and Their Prokaryotic Expression

作　　者：鹿连明[1] 林丽明[1] 谢荔岩[1] 林奇英[1] 吴祖建[1] 谢联辉[1] 

机构地区：[1]福建农林大学植物病毒研究所,福建省植物病毒学重点实验室,福州350002

出　　处：《农业生物技术学报》2008年第3期530-536,共7页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)(No.2006CB100203);国家自然科学基金(No.30671357);教育部博士点基金(No.20040389002);福建省自然科学基金(No.C0310010)资助

摘　　要：PCR扩增获得水稻(Oryza sativa L.ssp.japonica)叶绿体Rubisco SSU引导肽基因和水稻条纹病毒(Rice stripe virus,RSV)外壳蛋白(coat protein,CP)基因。分别借助于pGEX-4T-1和pET-29a表达载体,通过两种方法构建了融合基因PR-CP和PR-S-CP。测序表明,其读码框完整,连接部位正确。将重组子pGEX-PR-CP和pET-PR-S-CP转化大肠杆菌(Escherichia coli)BL21(DE3)并诱导表达。SDS-PAGE电泳检测其表达产物分子量分别为64和40kD,与预期结果大小一致。Western blot鉴定发现,两种融合基因的表达产物均能与RSV CP抗体特异结合,说明融合蛋白中RSV CP蛋白保持了自身的抗原活性,叶绿体Rubisco SSU引导肽并未影响其正确表达。可以推断两种融合基因PR-CP和PR-S-CP在生物体内表达时,融合蛋白中的Rubisco SSU引导肽和RSV CP蛋白可能都能保持各自独立的结构和功能。The coat protein （CP） gene of Rice stripe virus （RSV） and Rubisco SSU leader peptide gene office chloroplast were amplified by PCR. Depending on expression vector pGEX-4T-1 and pET-29a, fusion genes PR-CP and PR-S-CP were constructed by two methods. Sequencing results showed that the reading frames were full and ligation parts were correct. The recombinants pGEX-PR-CP and pET-PR-S-CP were transformed into Escherichia coli BL21 （DE3）, and the expression products of fusion genes were induced to express by SDS-PAGE electrophoresis and Western bolt. The results indicated that the molecular weights of two fusion proteins were 64 and 40 kD, respectively as expected, and the expression products could be specifically recognized by the antibody RSV CP. This suggested that RSV CP maintained its antigen activity, and its expression was not effected by Rubisco SSU leader peptide. The Rubisco SSU leader peptide and RSV CP in the fusion proteins may be maintained its own structure and function when the fusion genes PR-CP and PR-S-CP expressed in the organism.

关 键 词：水稻条纹病毒外壳蛋白 RUBISCO SSU引导肽 融合基因 原核表达 

分 类 号：S188[农业科学—农业基础科学]