文献类型：期刊文献

中文题名：美国白蛾核型多角体病毒ORF72原核表达载体的构建、表达及纯化

英文题名：Construction,Expression and Purification of Prokaryotic Expression Vector of Hyphantria cunea Nuclear Polyhedrosis Virus ORF72

作者：李娜[1] 李恩杰[2] 王青华[2] 王玉珠[2] 张永安[2] 段立清[1]

第一作者：李娜

机构：[1]内蒙古农业大学林学院;[2]中国林业科学研究院森林生态环境与保护研究所

年份：2018

卷号：31

期号：5

起止页码：57-63

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD2017_2018】；

基金：国家林业公益性行业科研专项"重大森林害虫持续防控关键技术与体系研究"(201504302)

语种：中文

中文关键词：美国白蛾核型多角体病毒;ORF72;载体构建;诱导表达;纯化

外文关键词：Hyphantria cunea nuclear polyhedrosis virus;ORF72;vector construction;induced expression;purification

分类号：S763.42

摘要：[目的]根据Ikdea对Hycu NPV全基因组序列的分析,筛选出功能还未被研究的、保守性高的ORF72基因。[方法]将ORF72基因构建到原核表达载体p GEX-4T-1上,对其进行诱导表达,优化表达的条件,并通过GST亲和层析色谱柱纯化融合蛋白。[结果]重组质粒p GEX-4T-1-ORF72的酶切检测以及测序结果均正确,证明重组质粒载体构建成功。通过SDS-PAGE凝胶电泳显示,ORF72蛋白能够与p GEX-4T-1载体上的GST标签蛋白进行融合表达,诱导表达后的融合蛋白大小约为38. 2 k Da。优化后的表达条件为:异丙基硫代β-D-半乳糖苷(IPTG)诱导浓度为1. 0 mmol·L-1,最佳诱导温度为25℃,最佳诱导时间为4 h,并且该蛋白在大肠杆菌BL21和Rosetta表达菌株中均能很好的表达,但在Rosetta菌株中表达量更高。[结论]成功构建了p GEX-4T-1-ORF72原核表达载体,确定出ORF72融合蛋白最佳诱导表达条件,诱导表达并纯化出ORF72融合蛋白,为进一步研究其生物功能奠定基础。
[Objective]The ORF72 gene of Hyphantria cunea nuclear polyhedrosis virus belongs to the GIY-YIG endonuclease family,it has a high conservative sequence and is closely related to the viral DNA replication. [Method]An ORF72 gene was sub-cloned into the p GEX-4 T-1 vector. The protein was over-expressed under different induced conditions and purified by GST-tag affinity chromatography column. [Result]It was proved that the recombinant vector was constructed successfully by the restriction map and DNA sequencing. SDS-PAGE gel electrophoresis detection showed that ORF72 protein could be integrated with GST-tag protein on the pGEX-4 T-1 and the size of the expressed fusion protein was about 38. 2 k Da. The induced conditions in over-expression of the recombinant proteins were optimized. The major recombinant protein was obtained by a feasible condition at 25℃ with 1. 0 mmol·L^-1 IPTG for 4 h. Moreover,the recombinant protein was expressed to high levels in the two strains of Escherichia coli BL21 and Rosetta,while the expression in the Rosetta strain was higher than BL21. [Conclusion]The recombinant ORF72 was purified using GST-affinity chromatography.