十二指肠贾第虫谷氨酸脱氢酶原核表达及动力学分析

Prokaryotic Expression and Dynamic Analysis of Glutamate Dehydrogenase in Giardia duodenalis

为探索十二指肠贾第虫(Giardia duodenalis)谷氨酸脱氢酶(glutamate dehydrogenase,GDH)在滋养体中的分布和酶动力学等生物学特性,对GDH进行了克隆并构建原核表达质粒pET-28a-GDH,然后转化至BL21(DE3)plysS感受态细胞中经IPTG诱导表达,对表达产物进行SDS-PAGE和Western blot分析,以Ni 2+亲和层析纯化。对纯化的重组GDH进行体外酶动力学分析并免疫小鼠制备多克隆抗体,最后用间接免疫荧光技术对滋养体中的GDH进行定位。结果显示,贾第虫GDH的编码序列长1350 bp,试验成功构建pET-28a-GDH重组表达质粒并诱导表达,重组蛋白约为49.7 ku,且大部分以可溶性形式存在。ELISA和Western blot结果表明,贾第虫GDH具有良好免疫原性,获得的多克隆抗体具有较强特异性。间接免疫荧光定位显示GDH主要位于滋养体的胞质中。酶动力学研究结果显示,GDH最适反应pH约为9.3、最适温度约为42℃,对L-谷氨酸和NADP+的Km值分别为4.485 mmol/L±0.5062 mmol/L和119.5μmol/L±10.1μmol/L。研究结果为深入研究贾第虫GDH的生物学功能奠定了基础。

To study subcellular localization and enzyme kinetics of glutamate dehydrogenase(GDH)in G.duodenalis trophozoites,the DNA encoding GDH was cloned to yield the prokaryotic expression vector pET-28a-GDH.The recombinant plasmid was transformed into E.coli BL21(DE3)plysS.The target protein was expressed with IPTG induction,validated by SDS-PAGE and Western blot,and purified by Ni 2+affinity chromatography.Enzyme kinetics of recombinant GDH was analyzed in vitro,and polyclonal antibodies against the recombinant GDH were prepared by immunization of mice.Finally,the GDH in trophozoites was localized by indirect immunofluorescence.The results showed that the coding region of G.duodenalis GDH was 1350 bp.The expression vector pET-28a-GDH was successfully constructed,and the recombinant GDH expression was efficiently induced.The recombinant GDH was 49.7 ku,most of which existed in a soluble form.ELISA and Western blot showed that GDH had considerable immunogenicity,and the polyclonal antibodies achieved high specificity.Indirect immunofluorescence assay showed that GDH was predominantly located in the cytoplasm of trophozoites.The optimal pH and temperature of GDH enzyme activity were 9.3 and 42℃,respectively.The GDH had Km values for L-glutamate and NADP+of 4.485 mmol/L±0.5062 mmol/L and 119.5μmol/L±10.1μmol/L,respectively.This study provided basis for further studies on the biological functions of G.duodenalis GDH.