家蚕核苷二磷酸激酶基因的克隆和原核表达

Cloning and Prokaryotic Expression of Bombyx mori Nucleoside Diphosphate Kinase Ecoding Gene

核苷二磷酸激酶(NDPK)基因在进化中高度保守,却又呈现复杂多样的生物学功能。该酶除了催化腺苷三磷酸(ATP)和核苷二磷酸(NDP)之间高能磷酸基团的转移外,还具有NDP激酶活性和蛋白磷酸转移酶活性,并参与转录调控和信号转导。从家蚕蛹cDNA文库中获得家蚕核苷二磷酸激酶的cDNA序列,该cDNA序列长575bp,含465 bp的开放阅读框序列,编码154个氨基酸残基的核苷二磷酸激酶。将克隆的家蚕核苷二磷酸激酶基因插入pET-28 a构建重组质粒,转化大肠杆菌后诱导表达重组蛋白,经镍离子亲和层析柱纯化得到重组家蚕核苷二磷酸激酶。

Nucleoside diphosphate kinase（NDPK） encoding genes are highly conservative in the evolution, but NDPKs reveal various biological funcions. Beside the catalysis in transferring the high energy phosphate bond from ATP to NDP,NDPKs also show the activities of NDP kinase and proteinic phosphotransferase. According to the large-scale sequencing of cDNA library from silkworm pupae, a cDNA which encodes Bombyx mori nucleoside diphosphate kinase （BmNDPK） was identified. This 575 bp cDNA sequence has a 465 bp open reading frame which encodes the 154 aa BmNDPK. The BmNDPK gene was cloned and then inserted into pET-28a to constructed a recormbinant plasmid pET-28a-BmNDPK. E. coil BL21 （DE3） was transformed with this recombinant plasmid and induced with IPTG to express the recombinant protein. Finally the recombinant BmNDPK was obtained by Ni^2＋ afifinity chromatography.