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首页> 外文期刊>Infection and immunity >Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica
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Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

机译：肝片Fasciola肝中含Cu / Zn的超氧化物歧化酶的分子克隆和表达

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The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.
机译：纯化并鉴定了筋膜炎的致病因子的胞质超氧化物歧化酶（SOD）。该酶由两个相同的亚基组成，每个亚基的表观分子量为17.5 kDa。对该酶的主要结构和抑制研究的分析表明，该酶是一种含铜/锌的SOD（Cu / Zn-SOD）。在pH 7.0至10.0的宽pH范围内，酶活性相对稳定，并且在pH 7.5时酶显示出最大活性。该酶还显示出对牛和筋膜炎患者血清的强抗原性。通过简并寡核苷酸引物通过PCR扩增SOD基因片段，所述简并寡核苷酸引物衍生自其他生物的Cu / Zn-SOD中保守的氨基酸序列。一个 F。以SOD基因片段为探针筛选肝cDNA文库。结果，鉴定出编码Cu / Zn-SOD的完整基因，并确定了其核苷酸序列。该基因具有438 bp的开放阅读框和146个推导氨基酸。该酶的推导氨基酸序列与先前报道的Cu / Zn-SOD氨基酸序列的比较显示出相当高的同源性。 F的编码区。克隆了肝/铜-超氧化物歧化酶并在大肠杆菌中表达。对表达的蛋白质的SOD活性进行染色的天然聚丙烯酰胺凝胶显示SOD活性被氰化钾和过氧化氢灭活，但没有被叠氮化钠灭活。这意味着重组融合蛋白的存在指示了Cu / Zn-SOD。表达的蛋白质还与带有筋膜病的牛和人血清反应，但不与未感染的牛和人血清反应。

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