家蚕浓核病毒镇江株非结构蛋白2(NS2)的表达及活性研究

Expression and Activity Analysis of Non-Structural Protein 2 (NS2) of Bombyx mori Densovirus Zhenjiang Strain

【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Westernblot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-ZNS2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0.276μmol·μg-1·h-1。【结论】BmDNV-ZNS2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。

[Objective] The non-structural protein 2 （NS2） gene ofBombyx 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.