蓝氏贾第鞭毛虫胞外核酸酶的表达纯化和活性鉴定

Prokaryotic expression and characterization of Giardia lambliaextracellular nuclease

目的克隆、原核表达蓝氏贾第鞭毛虫(Giardia lamblia,贾第虫)的胞外核酸酶编码区,并对其蛋白产物进行活性鉴定。方法对贾第虫胞外核酸酶(GeNuc)蛋白进行生物信息学分析,根据分析结果以C2株贾第虫基因组DNA为模板扩增获得GeNuc去信号肽段编码区序列,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。Ni-NTA亲和层析纯化GeNuc蛋白,经复性后验证其对质粒DNA的水解能力。结果成功克隆了长约800bp的GeNuc编码区并构建了原核表达载体pET-28a(+)-GeNuc,测序结果显示C2株GeNuc序列与WB株相同;在大肠杆菌中诱导表达获得了相对分子量约30.8kDa的融合蛋白;复性后的纯化GeNuc蛋白具有降解双链DNA的能力,但活性较商品化DNaseⅠ低。结论证明了GeNuc的存在,为GeNuc抗体的制备及贾第虫致病机制的研究提供了实验材料。

For many pathogens, extracellular nuclease is requisite for salvaging exogenous nucleosides and defending against immune attack. By searching the genome of Giardia larnblia, we found a provisional extracellular nuclease coding sequence. To express G. lambia extracellular nuclease （GeNuc） in E. coli and characterize its activity, the provisional GeNuc protein sequence of G. lambia WB strain from GenBank was analyzed. The coding sequence of GeNuc without signal peptide was amplified by PCR from genome DNA of G. lamblia C2 strain. Sequencing result showed that GeNuc in C2 strain was identical to that in Giardia WB strain. The PCR product （800 bp in size） was cloned into prokaryotic expression vector pET-28a（＋）. The recombinant vector pET-28a（＋）-GeNuc was transformed into E. coli Rosetta（DE3）, then the recombinant GeNuc protein was expressed by IPTG induction. SDS-PAGE and Western blot using anti-His Tag antibody showed that the expressed product of GeNuc was a fusion protein about 30.8 kD. The GeNuc recombinant protein, which was purified by Ni-NTA affinity chroma- tography and renatured by dialysis, showed DNase activity by partially digesting plasmid DNA. The successful prokaryotic ex- pression and characterization of GeNuc provide a prerequisite for antibody preparation and further approach of pathogenesis of Giardiasis.