鱼腥藻铁吸收调节蛋白基因(alr0957)的克隆和表达

Cloning and expressing of the fur gene(alr0957) from Anabaena sp. strain PCC 7120

依据CyanoBase提供的鱼腥藻PCC7120 furC基因(alr0957)的序列信息设计了一对特异性引物,用Touch-down PCR的方法从基因组DNA中扩增得到大小约450bp的目的片段.通过TA克隆的方法将该片段连接到pMD18-T载体上筛选出重组质粒pMD18-T-fur,然后进行双酶切,纯化furC基因,再连接到原核表达载体pET-28a(+)上,转化表达菌株BL21(DE3).经PCR、双酶切和测序鉴定,对阳性菌株进行IPTG诱导表达,SDS-PAGE检测重组蛋白.结果表明:在25℃条件下经1mmol/L IPTG诱导20h,融合蛋白被成功表达,其分子量约为19 000,为进一步纯化蛋白和对基因的调控功能方面研究奠定了基础.

In order to clone the fur gene from Anabaena sp.PCC 7120,we designed a pair of specific primers and amplified a segment about 450 bp by towch-down PCR from the Anabaena sp.PCC 7120 genome DNA,and then recombined the fragment to the plasmid pMD18-T,and then cloned the fur gene into prokaryotic expression vector pET-28a（＋） and transformed into E.coli strain BL21（DE3）.After PCR,restriction enzyme and sequencing analysis,The prokaryotic expression vector with target gene（pET-fur） was constructed successfully.Then,the fusion protein with a His-tag was expressed after induced by IPTG.SDS-PAGE showed the fusion protein about 19 000 was expressed in E coli.BL21.This research aims to purify the Fur protein for further and do functional verification.