柑橘碎叶病毒外壳蛋白基因的原核表达  

Prokaryotic expression of the CP gene of Citrus tatter leaf virus

作　　者：段硕[1,2] 李敏[1,2] 陶珍珍[1,2] 宋震 李中安 周常勇 

机构地区：[1]西南大学植物保护学院,重庆400716 [2]国家柑橘工程技术研究中心,重庆400712

出　　处：《果树学报》2013年第5期744-747,共4页Journal of Fruit Science

基　　金：重庆市自然科学基金(CSTC;2010BB1150);公益性(农业)行业专项(果树病毒病防控技术研究与示范;201203076);教育部创新团队(IRT0976)

摘　　要：【目的】获得原核表达的柑橘碎叶病毒(Citrus tatter leaf virus,CTLV)的外壳蛋白(Coat protein,CP)。【方法】以感染CTLV的柑橘叶片总RNA为模板,根据已报道CTLV设计引物,运用一步法RT-PCR对CTLV的CP进行扩增并克隆,并将CP克隆至表达载体pET28a(+),转化大肠杆菌BL21(DE3),经异丙基硫代半乳糖苷(IPTG)诱导表达获得表达蛋白。【结果】扩增测序结果显示,克隆产物CP区域序列全长714 bp,与已报道CP核苷酸序列(GenBank序列号:FJ223212)相似性达100%,推测编码238个氨基酸,表达蛋白的大小约27 kD。重组质粒经IPTG诱导表达后,SDS-PAGE分析结果显示,重组质粒高效表达约30 kD蛋白,酶联免疫吸附(Enzyme-linked immuno sorbent assay,ELISA)检测结果表明,该蛋白稀释5000倍仍能与苹果茎沟病毒(Apple stem grooving virus,ASGV)抗体发生阳性反应,证实表达蛋白为CTLV的CP。【结论】克隆了CTLV的CP,通过原核表达系统高效表达了CTLV的CP,为制备CTLV抗体奠定了基础。[Objective]The objective of the study was to obtain the coat protein of Citrus tatter leaf virus（CTLV） via the Prokaryotic Expression System. [Method]Total nucleic acids extracted from CTLV- infected trees were used as template for the amplification of CP gene by One-step reverse transcription PCR （RT-PCR） with a pair of specific primers. The fused expression plasmid was constructed by inserting the CP gene into the prokaryotic expression vector pET28a（＋） and then transformed into E. coli BL21 （DE3）. The recombined protein was expressed successfully after induced by isopropyl thiogalactoside （IPTG）. [ResultlSequence analysis showed that the length of CP gene was 714 base pairs, encoding 238 amino acid and expressing 27 kDa protein approximately. The sequenced CP gene showed 100% homology with the deposited CTLV CP gene （Accession No. at GenBank： FJ223212）. SDS-PAGE analysis indicated that an about 30 kDa expressed protein was obtained, which was consistent with that of expected protein. Positive reaction of expressed protein diluted 5 000 times with the Apple stem grooving virus antibody was observed by ELISA（enzyme-linked immuno sorbent assay）, suggesting the expressed protein was the coat protein of CTLV. [Conclusion]The CP gene of CTLV was successfully cloned and expressed in E. coli by Prokaryotic Expression System and the purified CP protein was suitable to the preparation of CTLV antibody.

关 键 词：柑橘碎叶病毒 CP 原核表达 蛋白纯化 

分 类 号：S666[农业科学—果树学]