田鼠巴贝斯虫醛酮还原酶BmAKR2的基因克隆和鉴定

Gene cloning and characterization of aldo-keto reductase BmAKR2 from Babesia microti

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。

In order to understand antioxidant ability of Babesia microti in oxidative stress environment,a novel aldo-keto reductase（AKR） gene was cloned from B.microti and identified.The full-length BmAKR2 cDNA contained an open reading frame of 2 490 bp in length,which encoded an 829 amino acid polypeptide.A classic AKRs conserved domain was found in the 172 nd to 542 nd amino acid residue by BLAST analysis.The conserved domain was subcloned into the pGEX-4 T-1 expression vector,and the protein was expressed as a glutathione S-transferase（GST）-fusion protein in Escherichia coli BL21（DE3） cells.Recombinant BmAKR2-HX protein was injected into mice to produce antibodies.The result of Western-blot showed a native BmAKR weight was approximately 96 ku that was similar to the predicted value.The result of indirect immunofluorescence assay showed that BmAKR2 was located in the nuclei of B.microti merozoites.The results of real time RT-PCR showed BmAKR2 mRNA was transcribed on 2-14 days post infection（DPI）,but was highest on 5 DPI.