C2株蓝氏贾第鞭毛虫TCTP基因的克隆、表达和序列分析（英文）

Cloning,prokaryotic expression and sequencing of TCTP gene of Giardia lamblia C2 strain

目的克隆C2株蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)的翻译调控肿瘤蛋白(Translationally controlled tumor protein,TCTP)编码区,对其进行生物信息学分析和原核表达。方法根据WB株贾第虫TCTP编码区序列设计引物,以C2株贾第虫基因组DNA为模板扩增获得TCTP编码区片段,双酶切连入原核表达载体pET-28a(+),将酶切和测序验证正确的重组质粒转化E.coli Rosetta(DE3),经IPTG诱导表达融合蛋白,SDS-PAGE及Western blot鉴定蛋白产物,采用Ni-NTA经亲和层析纯化重组蛋白。同时根据测序结果分析C2株贾第虫TCTP蛋白结构特征和进化关系。结果克隆了包含酶切位点全长470bp的C2株贾第虫TCTP编码区,构建了原核表达载体pET-28a(+)-TCTP,在Rosetta(DE3)中表达、纯化得到了相对分子量约18.5kDa的融合蛋白,与预期相符;测序结果显示C2株贾第虫TCTP序列与WB株相同,生物信息学分析显示贾第虫TCTP蛋白虽然序列长度较其它物种更短但与裂殖酵母TCTP空间结构相似,均由4个β折叠和3个主要的α螺旋组成,位于序列中部的不规则环状结构更小,仅9个氨基酸残基组成,在进化上与其它真核生物亲缘关系较远。结论成功克隆表达了贾第虫TCTP,为TCTP功能及贾第虫致病机制的研究提供了实验依据。

Translationally controlled tumor protein （TCTP） is a highly conserved muhifunctional protein present in all eu- karyotic organisms. Recent researches showed that TCTP was involved in pathogenesis of some hematologic parasites. In order to express Giardia lambia TCTP （G1TCTP） in E. coli, the full-length open reading frame of TCTP was amplified by PCR from Giardia lamblia （C2 strain） genome DNA. The PCR product about 470 bp in length was cloned into prokaryotic expres- sion vector pET-28a（＋） with restriction enzymes Nco I and Xho I. The recombinant vector pET-28a（ ＋）-TCTP was trans- formed into E. coli Rosetta （DE3） and expressed by IPTG induction. The TCTP fusion protein about 18.5 kDa was detected by SDS-PAGE and Western blotting and purified by Ni-NTA affinity chromatography. The resuh revealed that the sequence of TCTP in C2 strain was identical with that in WB strain. Bioinformatics analysis showed that Giardia TCTP protein was shor- ter than that of other eukaryotes, but had a similar structure with Schizosaccharomyces pombe TCTP. The irregular ring struc- ture in the central region of G1TCTP, which consisted of 9 residues, was smaller than that of SpTCTP. Sequence alignment of TCTP revealed a distant relationship between G lamblia and other eukaryotes. The successful prokaryotic expression and bioin- formatics analysis of Giardia TCTP provide information for the study of pathogenic mechanism of G. lamblia and an ideal mod- el organism for TCTP approach.