家蚕尿酸氧化酶基因BmUo的克隆及其序列分析与原核表达

Cloning and Sequence Analysis of Urate Oxidase Gene in the Silkworm,Bombyx mori,and its Expression in Prokaryotic System

尿酸氧化酶(urate oxidase;EC1.7.3.3)能够降解尿酸形成尿囊素,在嘌呤代谢途径中起到至关重要的作用。用生物信息学方法在家蚕基因组数据库中进行同源性检索,获得一条可能的家蚕尿酸氧化酶序列,根据预测序列设计引物及克隆、测序验证,已成功地克隆到家蚕尿酸氧化酶基因BmUo的完整ORF。克隆的cDNA(GenBank登录号:DQ189992)全长1088bp,ORF长1014bp,编码337个氨基酸残基,预测分子质量为38.18kD,等电点为6.90。通过半定量RT-PCR检测了该基因在各组织的表达情况,发现该基因的mRNA在5龄第3天的家蚕的脂肪体和马氏管中有表达。该基因在氨基酸水平上与果蝇、按蚊尿酸氧化酶的相似性分别为45.6%和51%,在第91个氨基酸残基处具有Asn-Ser-X-Val/Ile-Val/Ile-Ala/Pro-Thr-Asp-Ser/Thr-X-Lys-Asn的高度保守序列,这一序列在真核生物和原核生物中均广泛存在,可能与尿酸氧化酶的酶活性有关。家蚕尿酸氧化酶与其他38个物种尿酸氧化酶的聚类分析发现,尿酸氧化酶基因在昆虫、真菌、双子叶植物和哺乳动物这一层面上是相对保守的。将BmUo基因片段与pET-28a连接,构建原核表达载体pET-28a/uo,重组载体转化大肠杆菌BL21后,在37℃的培养条件下可检测到与理论分子质量相符的蛋白条带。

The urate oxidase plays an important role in the purine metabolism. The animo acid sequence of Dro- sophila DrUo was searched against the silkworm genome database with BLAST program. The CDS with high score were clustered. By using the primers based on the consensus sequence, we cloned the urate oxidase gene of Bom- byx mori, termed it as BmUo. The length of the BmUo cDNA was 1088 base pairs, which encoded 337 amino acids, with the predicted molecular weight of 38. 18 kD and isoelectric point of 6. 90. The deduced amino acid sequence of the BmUo shared 45. 6% identity with Drosophila DrUo and 51% with A. gambiae Uo. There was a high conserved region Asn-Ser-X-Val/lle-Val/lle-Ala/Pro-Thr-Asp-Ser/Thr-X-Lys-Asn in BmUo, which maybe related with activity of urate oxidase. A clustering analysis showed that the BmUo was classified with urate oxidase of Drosophila melanogaster and Apheles gambiae. Using pET-28a as the expression vector, a recombinant plasmid pET- 28a/uo was constructed. Induced by IPTG in 37℃, the target protein was detected.