中文摘要：

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。

英文摘要：

To clone,express and identify the biliverdin reductase（BVR） eDNA of Yanbian yellow cattle in E. coll. The primers were designed according to cDNA sequence of the BVR gene of Bos taurus ,Equus caballus and Panthera tigris altaica and the full cDNA sequence of the BVR gene of Yanbian yellow cattle was obtained by 3＇ and 5＇ RACE. The recombinant plasmid of pET -28a- BVR was constructed,transformed into BL21 E. coli （DE3） and purified. The enzyme activity of BVR was detected by spectrophotometry. The cDNA reading framesequence of the BVR gene of Yanbian yellow cattle covers 891 bases, encoding 296 amino acids. The homology analysis by BLAST and ClusalW software showed that the deduced nucleotides of Yanbian yellow cattle BVR shared 98.0%,88.0% and 87.0% identity with those of Bos taurus,Equus caballus and Panthera tigris altaica, respectively, the molecular weight of BVR proteins, as measured by using SDS-PAGE was 32 560. The catalytic activity determination showed that the purified proteins could catalyze the reduction of biliverdin to bilirubin. The active BVR enzyme protein of Yanbian yellow cattle was obtained by cloning and expression,which provides a basis of the further preparation of biliru- bin.