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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。
Objective To clone and express the extracellular nuclease coding region of Giardia lamblia (Giardia lamblia), and to identify its activity. Methods Bioinformatics analysis of Giardia exocytase (GeNuc) protein was carried out. According to the results of analysis, genomic DNA of Giardia was used as a template to amplify the GeNuc signal peptide coding region. The recombinant plasmid pET-28a (+) was transformed into E.coli Rosetta (DE3). The fusion protein was induced by IPTG. The protein product was identified by SDS-PAGE and Western blot. The GeNuc protein was purified by Ni-NTA affinity chromatography and verified by its refolding ability to hydrolyze plasmid DNA. Results The GeNuc coding region of about 800 bp was successfully cloned and the prokaryotic expression vector pET-28a (+) - GeNuc was successfully cloned. The sequencing results showed that the GeNuc sequence of C2 strain was the same as that of WB strain. The relative molecular weight was about 30.8 kDa fusion protein; Refolded purified GeNuc protein has the ability to degrade double-stranded DNA, but its activity is lower than that of commercial DNaseⅠ. Conclusion The existence of GeNuc is proved, which provides the experimental materials for the preparation of GeNuc antibody and the pathogenesis of Giardia.