延边黄牛胆绿素还原酶cDNA的克

发布时间：2018-05-08 23:42

本文选题：延边黄牛 + 胆绿素还原酶　；参考：《中国兽医学报》2017年05期

【摘要】：为了克隆延边黄牛胆绿素还原酶(Biliverdin reductase,BVR)的CDNA序列后在大肠杆菌中表达、纯化并进行鉴定。本试验根据黄牛、马和东北虎BVR基因的cDNA序列设计引物,通过3′和5′RACE方法获得延边黄牛BVR基因全长cDNA序列。之后构建pET-28a-BVR重组质粒并转化到E.coli BL21(DE3)中表达并进行纯化。通过分光光度法检测BVR酶的活性。结果显示,所获得的延边黄牛BVR基因cDNA含有1个完整的开放阅读框,该cDNA序列阅读框由891个核苷酸组成,编码296个氨基酸。用BLAST和ClustalW软件进行同源性分析,与牛、马和东北虎的核苷酸相似性分别为98.0%,88.0%和87.0%。SDS-PAGE鉴定其相对分子质量为32 560。分光光度法检测BVR具有催化胆绿素还原为胆红素的活性。结果表明,通过克隆表达获得了有活性的延边黄牛BVR酶蛋白,为胆红素的进一步大量制备提供理论基础。
[Abstract]:In order to clone the CDNA sequence of Biliverreductin reductase (Biliverreductase BVR) from Yanbian cattle, it was expressed in Escherichia coli, purified and identified. Primers were designed according to the cDNA sequence of BVR gene of yellow cattle, horse and Siberian tiger, and the full-length cDNA sequence of BVR gene of Yanbian cattle was obtained by 3 'and 5'RACE methods. PET-28a-BVR recombinant plasmid was then constructed and transformed into E.coli BL21 (DE3) for expression and purification. The activity of BVR enzyme was detected by spectrophotometry. The results showed that the BVR gene cDNA of Yanbian cattle contained a complete open reading frame, which consisted of 891 nucleotides and encoded 296 amino acids. The nucleotide similarity with cattle, horse and Amur tiger was 98.0% and 88.0%, respectively, and the relative molecular weight of Amur tiger was 32560 by using BLAST and ClustalW software. BVR has catalytic activity of reducing bilirubin to bilirubin by spectrophotometry. The results showed that the active BVR protein of Yanbian cattle was obtained by cloning and expression, which provided a theoretical basis for the further preparation of bilirubin.
【作者单位】：延边大学农学院;
【基金】：国家自然科学基金资助项目(31160501)
【分类号】：S823.81

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