番木瓜畸形花叶病毒HC-Pro蛋白的原核表达与纯化
Prokaryotic Expression and Purification of HC-Pro Protein of Papaya Leaf Distortion Mosaic Virus
苏蝶 1庹德财 2周鹏 1言普 2黎小瑛 2朱国鹏 3沈文涛1
作者信息
- 1. 海南大学园艺学院,海口,570228;中国热带农业科学院热带生物技术研究所,海口,571101
- 2. 中国热带农业科学院热带生物技术研究所,海口,571101
- 3. 海南大学园艺学院,海口,570228
- 折叠
摘要
番木瓜畸形花叶病毒(papaya leaf distortion mosaic virus,PLDMV,Potyvirus属)是目前威胁中国番木瓜种植的主要病原之一.辅助成分-蛋白酶(helper component-proteinase,HC-Pro)是PLDMV编码参与病毒运动、复制、媒介传播、基因沉默抑制的多功能蛋白.为了获得HC-Pro基因的纯化蛋白,本研究通过密码子优化HC-Pro基因序列,成功构建原核表达质粒pET-30a-HCPro,经IPTG诱导后实现HC-Pro在大肠杆菌中的原核表达,在0.5 mmol/L IPTG,20 ℃条件下诱导过夜时其表达量最高;破碎菌体后,SDS分析全菌、破碎上清以及沉淀中目标蛋白的表达量,结果显示重组HC-Pro蛋白主要以不可溶的包涵体形式在沉淀中表达;进一步溶解包涵体通过Ni-NTA层析柱进行纯化,在500 mmol/L浓度咪唑下可以获得分子量大小约为54 kD的HC-Pro重组蛋白;Western Blot分析纯化后的目标蛋白,结果显示54 kD处有明显的条带,与预期的HC-Pro重组蛋白大小一致.本研究成功表达并纯化获得了 PLDMV重组蛋白HC-Pro,为后续抗体制备和探究其功能提供了理论基础.
Abstract
Papaya leaf distortion mosaic virus(PLDMV,genus Potyvirus)is becoming a new major limiting factor for the economical production of papaya in China.Helper component-proteinase(HC-Pro)is a potyviral multi-functional protein involved in virus movement,replication,vector transmission,and RNA silencing.In order to ob-tain the purified protein of HC-Pro gene,this study successfully constructed the prokaryotic expression plasmid pET-30a-HCPro by codon optimization of HC-Pro gene sequence;the highest expression of HC-Pro in E.coli was achieved after induction with IPTG and was induced overnight at 0.5 mmol/L IPTG at 20 ℃;after fragmentation,SDS analysis of the expression of target proteins in whole bacteria,fragmented supernatants and precipitates showed that the recombinant HC-Pro protein was mainly expressed in the precipitates as insoluble inclusion bodies;further purification of the solubilized inclusion bodies by means of Ni-NTA chromatography columns resulted inHC-Pro re-combinant protein with a molecular weight size of approximately 54 kD at a concentration of500 mmol/L imidazole;western Blot analysis of the purified target protein showed a clear band at 54 kD,consistent with the expected size of the HC-Pro recombinant protein.In this study,PLDMV recombinant protein HC-Pro was successfully expressed and purified,which provided a theoretical basis for subsequent antibody preparation and investigation of its function.
关键词
番木瓜畸形花叶病毒/HC-Pro/原核表达/纯化Key words
Papaya leaf distortion mosaic virus/HC-Pro/Prokaryotic expression/Purification引用本文复制引用
基金项目
海南省基础与应用基础研究计划(自然科学领域)自然科学领域高层次人才项目(2019RC298)
国家自然科学基金项目(32072390)
出版年
2024
NETL
NSTL
万方数据