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| 家蚕浓核病毒镇江株非结构蛋白2(NS2)的表达及活性研究 | |
| 引用本文： | 赵盼,唐顺明,刘挺,覃光星,郭锡杰.家蚕浓核病毒镇江株非结构蛋白2(NS2)的表达及活性研究[J].中国农业科学,2009,42(6):2149-2155. |
| 作者姓名： | 赵盼 唐顺明 刘挺 覃光星 郭锡杰 |
| 作者单位： | 1. 江苏科技大学生物与环境工程学院,江苏镇江,212003 2. 江苏科技大学生物与环境工程学院,江苏镇江,212003;中国农业科学院蚕业研究所/农业部家蚕生物技术重点开放实验室,江苏镇江,212018 3. 中国农业科学院蚕业研究所/农业部家蚕生物技术重点开放实验室,江苏镇江,212018 |
| 基金项目： | 国家重点基础研究发展规划(973计划) |
| 摘 要： | 【目的】对家蚕浓核病毒镇江株非结构蛋白2（NS2）基因进行克隆、表达,并测定表达产物的生物活性。【方法】利用PCR技术从家蚕浓核病毒镇江株（BmDNV-Z）基因组中扩增得到非结构蛋白2(NS2)基因片段,将其克隆到表达载体pET28a得到重组表达质粒pET28a-NS2,在大肠杆菌BL21（DE3）中进行表达,SDS-PAGE和Western blot检测,表达产物经Ni柱纯化,经过复性检测其Helicase和ATPase活性。【结果】克隆获得了BmDNV-Z NS2基因,并在大肠杆菌中得到了成功表达,表达产物经Ni柱纯化获得了目的蛋白NS2。纯化的NS2蛋白具有Helicase活性,能将双链DNA底物解旋成为单链,并且具有一定的底物极性选择性,对于极性底物表现出更高的解旋活性。同时,纯化的NS2蛋白具有ATPase活性,其酶活力可达到0.276 μmol?μg-1?h-1。【结论】BmDNV-Z NS2基因编码的病毒非结构蛋白具有Helicase和ATPase活性,并且Helicase活性具有一定的底物极性选择性,推测该基因在病毒DNA的复制过程中发挥重要作用。 |
| 关 键 词： | 家蚕 浓核病毒 非结构蛋白 表达 活性 |
| 收稿时间： | 2008-8-20 |
Expression and Activity Analysis of Non-Structural Protein 2(NS2)of Bombyx mori Densovirus Zhenjiang Strain |
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| ZHAO Pan,TANG Shun-ming,LIU Ting,QIN Guang-xing,GUO Xi-jie.Expression and Activity Analysis of Non-Structural Protein 2(NS2)of Bombyx mori Densovirus Zhenjiang Strain[J].Scientia Agricultura Sinica,2009,42(6):2149-2155. | |
| Authors: | ZHAO Pan TANG Shun-ming LIU Ting QIN Guang-xing GUO Xi-jie |
| Institution: | (College of Biotechnology and Environmental Engineering, Jiangsu University of Science and Technology) |
| Abstract: | 【Objective】 The non-structural protein 2 (NS2) gene of Bombyx mori densovirus Zhenjiang strain (BmDNV-Z) was cloned and expressed so as to test the biological activities of the expressed recombinant protein. 【Method】 The gene of the non-structure protein 2 (NS2) was cloned by PCR from the genome of Bombyx mori densovirus Zhenjiang strain, inserted into prokaryotic expression vector pET28a to construct recombinant plasmid pET28a-NS2 and then expressed in bacteria E. coli BL21 (DE3). The expressed recombinant protein was identified by SDS-PAGE and Western blot analysis. Then the recombinant protein was purified by Ni-NTA column, renatured and tested for enzyme activities. 【Result】 The gene of BmDNV-Z NS2 was cloned and successfully expressed in bacteria E. coli, and the expressed target protein NS2 was purified by Ni–NTA affinity chromatography. The purified NS2 protein exhibited a helicase activity unwinding double-stranded DNA substrates into single-strand primers, and higher unwinding activity to polarity substrate. At the meantime, the purified NS2 protein possessed a ATPase activity and its enzyme activity was 0.276 μmol?μg-1?h-1 in this study. 【Conclusion】 The non-structure protein which encoded by the gene of BmDNV-Z NS2 possesses the biological activities of helicase and ATPase, and meanwhile the helicase prefers to polarity substrates. Based on these results, it is speculated that the gene of BmDNV-Z NS2 plays an important role in the viral DNA replication. |
| Keywords: | Bombyx mori densovirus non-structure protein expression activity |
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