肺炎支原体铁吸收调节蛋白基因的克隆表达与纯化  

Cloning,expression and purification of Fur from Mycoplasma pneumoniae in E. coli

作　　者：唐愈菲[1,2] 游晓星[2] 赵爽[1] 黄泽智[1] 李冉辉[2] 

机构地区：[1]邵阳医学高等专科学校,湖南邵阳422002 [2]南华大学病原生物学研究所特殊病原体防控湖南省重点实验室,湖南衡阳421001

出　　处：《生物技术》2016年第3期219-223,251,共6页Biotechnology

基　　金：湖南省科技计划项目("次抑菌浓度的大环内酯药物诱导Mp交叉耐药的机制研究";No.2013FJ3067);特殊病原体防控湖南省重点实验室资助项目(湘科计字[2014]10号)

摘　　要：[目的]克隆肺炎支原体铁吸收调节蛋白(Fur)基因并纯化Fur蛋白,为研究其生物学功能奠定基础。[方法]利用Clustal Omega分析肺炎支原体Fur蛋白及其同源序列并用MEGA6.0构建进化树,通过PCR扩增Fur基因并对其进行双酶切,然后连接到p ET28a,得到重组载体p ET28a-Fur,将其转化大肠杆菌BL21(DE3),利用IPTG诱导Fur蛋白表达,并通过亲和层析纯化Fur蛋白。[结果]多序列比对和进化树分析表明Mp含有一个Fur蛋白。克隆得到大小为477 bp的Fur基因,编码159个氨基酸。得到重组表达质粒p ET28a-Fur,该质粒能在大肠杆菌中能高效表达,并纯化得到重组Fur蛋白。[结论]成功克隆获得Mp Fur基因,在大肠杆菌中高效表达并获得高纯度Fur蛋白。[ Objective ] To clone Fur （ Ferric uptake regulator protein） gene form Mycoplasma pneumoniae （Mp） and purify Fur protein. [ Methods ] The Mp Fur with its homologous proteins were analysed by Clustal Omega and the phylogenetic tree was constructed by MEGA6.0. The Mp Fur was amplified by PCR and ligated pET28a vector to generate the pET2$a - Fur plasmid, which was transformed into E. coli BI21 （ DE3 ）. The Fur protein was induced by IPTG and purified by Ni2＋ - affinity chromatography. [ Results ] The results of sequence alignment and phylogenetic tree show Mp possesses a Fur protein. The Mp Fur gene, which has 477 bp and encodes 159 amino acids, is inserted into pET28a, resulting in the recombinant plasmid pET28a - Fur. The over - expressed His - tagged Fur protein was purified successfully by Ni2＋ - affinity chromatography. [ Conclusion] The Fur from Mp was successfully cloned and expressed in E. coli. The purified His -tagged Fur protein is obtained, which provides a foundation for further study on Fur.

关 键 词：肺炎支原体 Fur蛋白 克隆 表达 

分 类 号：Q936[生物学—微生物学]