C2株蓝氏贾第鞭毛虫SUMO基因的克隆、原核表达和生物信息学分析(英文)

Cloning,prokaryotic expression and bioinformatics analysis of Giardia lamblia C2 strain SUMO gene

目的克隆、原核表达C2株蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)的小泛素相关修饰物(small ubiquitn-related modifier,SUMO)蛋白,并对其序列进行生物信息学分析。方法提取C2株贾第虫基因组DNA,以基因组DNA为模板PCR获得SUMO基因片段,双酶切连入原核表达载体pET-28a(+),经测序验证并进行生物信息学分析,将重组质粒pET-28a(+)-SUMO转化大肠杆菌Rosetta(DE3)。IPTG诱导后收集菌体,裂解后进行SDS-PAGE及Western blot检测。结果成功构建了C2株贾第虫SUMO基因原核表达载体pET-28a(+)-SUMO,经IPTG诱导后,在大肠杆菌中高效表达,SDSPAGE及Western blot分析显示,在相对分子量约12.7kD的位置出现目的蛋白条带,与理论值相符;生物信息学分析显示C2株贾第虫SUMO序列与WB株相同,其蛋白形成类似"泛素折叠"样结构,进化分析表明贾第虫SUMO蛋白与其它现存真核生物亲缘关系较远。结论成功地克隆、表达了贾第虫SUMO蛋白,明确了该蛋白的空间结构和进化地位,为贾第虫SUMO蛋白抗体的制备及相关蛋白功能的研究提供了基础。

Small ubiquitin-like modifier （or SUM（）） proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. In order to express Giardia lamblia （C2 strain） SUMO pro tein in E. coli, the full-length open reading frame of SUM（） was ampIified by PCR from Giardia lamblia genome DNA. The PCR product about 320 bp in length was cloned into prokaryotic expression vector pET-28a（ ＋ ） with restriction enzymes Nco I and Xho I. Sequencing result showed the sequence of SUMO in C2 strain was identical with that in WB strain. Bioinformatics analysis showed that SUMO protein displayed an ubiquitin-fold structure in three-dimensional space. Phylogenetic analysis showed a distant relationship of SUMO protein between Giardia lamblia and other eukaryotes. The recombinant vector pET-28a（＋）-SUMO was transformed into E. coli Rosetta（DE3）, then the recombinant SUMO protein was expressed by IPTG induction. SDS-PAGE and western blot using anti-His Tag antibody showed that the expression product of SUMO was a fusion protein about 12.7 kD. The successful prokaryotic expression and bioinformatics analysis of Giardia lamblia SUMO protein provide basis for antibody preparation and functional study of SUMO related protein.