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为了解田鼠巴贝斯虫(Babesia microti)在富氧环境下的抗氧化能力,克隆了田鼠巴贝斯虫醛酮还原酶(aldo-keto reductases,AKRs)基因并进行了鉴定。全长的田鼠巴贝斯虫醛酮还原酶(BmAKR2)包含2 490 bp的阅读框,编码829个氨基酸。经BLAST分析,发现该蛋白质在第172~542位氨基酸残基区域存在典型的AKR结构域。将该酶的结构域克隆到表达质粒pGEX-4T-1中,然后转化到BL21(DE3)中进行原核表达,获得了GST融合重组蛋白GST-BmAKR2-HX。使用该重组蛋白免疫小鼠制备抗体,Western-blot鉴定结果显示,天然BmAKR2蛋白的分子质量为96 ku,与预测的大小一致。间接免疫荧光试验结果显示BmAKR2定位于田鼠巴贝斯虫裂殖子的细胞核。通过实时荧光定量PCR分析,表明BmAKR2在感染小鼠的第2~14天内均表达,其中第5天为表达峰期。
To understand the antioxidant capacity of Babesia microti in oxygen-rich environment, we cloned and identified the aldo-keto reductases (AKRs) gene of voles. The full-length vole Babesia insect aldehyde ketoreductase (BmAKR2) contains a 2 490 bp reading frame encoding 829 amino acids. After BLAST analysis, it was found that there was a typical AKR domain in the region of amino acid residues 172-542. The domain of the enzyme was cloned into the expression plasmid pGEX-4T-1 and transformed into BL21 (DE3) for prokaryotic expression. GST fusion recombinant protein GST-BmAKR2-HX was obtained. The recombinant protein was used to immunize mice to prepare antibodies. The results of Western-blot showed that the molecular weight of native BmAKR2 protein was 96 ku, consistent with the predicted size. Indirect immunofluorescence assay showed that BmAKR2 was localized in the nucleus of Babesia melligenel. Real-time quantitative PCR analysis showed that BmAKR2 was expressed in the 2nd to 14th days of infected mice, of which the 5th day was the peak period of expression.