C2株贾第虫醛糖还原酶的原核表达与生物信息学分析

Prokaryotic expression and bioinformatics analysis of Giardia lamblia C2 strain aldose reductase gene

[目的]克隆、原核表达、纯化C2株蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)醛糖还原酶(Aldose reductase,AR)基因,并进行生物信息学分析。[方法]从C2株贾第虫基因组DNA中克隆贾第虫AR编码区,双酶切连入原核表达载体pET-28a(+),酶切和测序进行验证,并进行生物信息学分析;将构建成功的重组质粒pET-28a(+)-AR转化大肠杆菌Rosetta(DE3)并进行诱导表达,SDS-PAGE及Western Blot验证表达效果;镍亲和层析纯化AR蛋白,SDS-PAGE观察纯化效果。[结果]成功克隆了C2株贾第虫AR蛋白编码区并构建了原核表达载体,SDS-PAGE及Western Blot显示,AR表达载体转化的大肠杆菌经诱导可表达出相对分子量约37.2k Da的目的蛋白,与预期一致;重组蛋白通过亲和层析获得了高效纯化;生物信息学分析显示贾第虫AR蛋白主要二级结构为无规则卷曲,整体为一个NADPH依赖的氧化还原酶结构域。AR蛋白在真核生物中相当保守,贾第虫AR蛋白在进化上相对独立。[结论]证实了贾第虫AR蛋白的存在并明确了其结构和进化上的特征,为贾第虫AR抗体的制备和功能的研究提供了基础。

[Objective]To express Giardia lambia AR protein prokaryotically,and analyze its by bioinformatics.[Methods]The coding sequence of AR was obtained by PCR from Giardia lamblia（C2 strain） genome DNA and inserted into prokaryotic expression vector pET-28a（＋） by double digestion.The recombinant vector was transformed into E.coli Rosetta（DE3） and expressed by induction of IPTG.The recombinant protein was identified by SDS-PAGE and Western Blot using anti-His tag antibody,and purified by Ni-NTA affinity chromatography.The amino acid sequence of AR was analyzed by many bioinformatics softwares.[Results] Restriction analysis and sequencing showed that recombinant vector pET28a（＋）-AR was constructed successfully.The recombinant AR protein,with a relative molecular weight about 37.2 kDa,was visualized by SDS-PAGE and Western Blot.Bioinformatics analysis suggested that the main secondary structure of Giardia AR protein was random coil and the whole length protein constituted a NADPH-dependent oxidoreductase domain.Evolutionary analysis showed that AR protein was highly conserved in eukaryotes and Giardia AR protein was relatively independent.[Conclusion] The successful prokaryotic expression and bioinformatics analysis of Giardia AR protein provide basis for antibody preparation and function study of AR.