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当前位置: 首页 > 文章 > 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 林业科学研究 2018 (5) 57-63
Position: Home > Articles > Construction,Expression and Purification of Prokaryotic Expression Vector of Hyphantria cunea Nuclear Polyhedrosis Virus ORF72 Forest Research 2018 (5) 57-63

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

作  者：
李娜;李恩杰;王青华;王玉珠;张永安;段立清
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
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。
译  名：
Construction,Expression and Purification of Prokaryotic Expression Vector of Hyphantria cunea Nuclear Polyhedrosis Virus ORF72
作  者：
LI Na;LI En-jie;WANG Qing-hua;WANG Yu-zhu;ZHANG Yong-an;DUAN Li-qing;Forestry College,Inner Mongolia Agricultural University;Research Institute of Forest Ecology,Environment and Protection,Chinese Academy of Forestry;
单  位：
LI Na%LI En-jie%WANG Qing-hua%WANG Yu-zhu%ZHANG Yong-an%DUAN Li-qing%Forestry College,Inner Mongolia Agricultural University%Research Institute of Forest Ecology,Environment and Protection,Chinese Academy of Forestry
关键词：
Hyphantria cunea nuclear polyhedrosis virus;;ORF72;;vector construction;;induced expression;;purification
摘  要：
[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.

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