家蚕浓核病毒(伊那株)(Bombyx Mori Densovirus,BmDNV-1)非结构蛋白基因NS2的克隆与表达

Cloning and Expressing of Nonstructural Protein Gene NS2 of Bombyx Mori Densovisus(BmDNV-1)

本文对家蚕浓核病毒伊那株的非结构基因NS2进行了克隆和表达,以期进一步研究其功能。以伊那株的全基因组为模板,PCR扩增出NS2基因,将其克隆入表达载体pET-28a(+)中,转化到DH5a感受态细胞中培养筛选阳性克隆pET-28a-NS2,并抽提质粒进行验证。重组质粒经BamHI和HindIII双酶切和测序证实正确,然后转化到表达菌株大肠杆菌BL21(DE3)中。用IPTG诱导表达NS2融合蛋白,通过10%SDS-PAGE检测表明:经1mMIPTG诱导4h后,目的蛋白的表达量最大,得到的NS2融合蛋白约56.0KD,并经Western blot进一步验证正确。

The nonstructural protein gene NS2 of Bombyx mori densovisus(BmDNV-1) was cloned and expressed to study its function.NS2 gene was amplified by PCR from the infectious clone of BmDNV-1 and inserted into the prokaryotic expression vector pET-28a(+).The recombinant plasmid pET-28a-NS2 was transformed into the competent cell DH5α and the correct recombinant clone was confirmed by BamHI and HindIII digestion and sequencing.Then the plasmid pET-28a-NS2 was transferred to BL21(DE3) to express the fusion protein NS2 by induction with IPTG.The result showed that the highest expression of the fusion protein was reached after 4 hours of the induction with 1mM IPTG.The NS2 fusion protein with the size of 56.0kDa was revealed by Western blot by using antibody against His·tag which is consistent with the expected size of the fusion protein.