西方蜜蜂DHPR基因的克隆、序列分析及原核表达
首发时间:2020-09-15
摘要:研究西方蜜蜂四氢生物蝶呤合成途径中的关键酶二氢蝶呤还原酶的功能,并探讨其对于蜜蜂中四氢生物蝶呤水平和线粒体结构的维稳作用。利用 RT-PCR方法,以西方蜜蜂的 cDNA 为模板扩增西方蜜蜂 DHPR 基因编码区,并通过软件 MEGA5.1 构建系统发育树,运用Psipred和 SWISS-MODEL 等网站预测分析蛋白结构; 用大肠杆菌 Escherichia coli 表达系统进行原核表达; 采用镍琼脂糖亲和层析法进行重组蛋白纯化。经克隆得到西方蜜蜂DHPR基因编码区长为 714 bp,命名为AmDhpr。该基因编码 237 个氨基酸,预测该编码蛋白的等电点为 8.90。系统进化树分析表明,西方蜜蜂AmDhpr与大蜜蜂和小蜜蜂的Dhpr聚成一支。蛋白质二级结构预测发现其含有 8 个 α-螺旋区和 8 个 β-折叠区。利用同源建模获得蛋白质的三维结构。将克隆得到的AmDhpr的编码区全长序列连接到 PET-30a 表达载体上,进一步转化到BL21(DE3)大肠杆菌感受态细胞,后培养并诱导表达,最后纯化获得大小约为25 kD的重组 DHPR 蛋白,与预期大小一致。本研究克隆了蜜蜂DHPR基因AmDhpr,并对其序列进行了分析,获得了纯化的重组蛋白,为进一步研究该基因的功能提供了参考和材料。
关键词: 西方蜜蜂; 二氢蝶呤还原酶; 四氢生物蝶呤; 序列分析; 结构预测; 原核表达
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Molecular Cloning, Sequence Analysis and Prokaryotic Expression of DHPR Gene in Apis Mellifera.
Abstract:In order to study the function of dihydropterin reductase, a key enzyme in the synthesis of tetrahydrobiopterin in western honeybees, and its effect on the maintenance of tetrahydrobiopterin level and mitochondrial structure in honeybees was investigated. The cDNA sequence of the DHPR gene was cloned from Apis Mellifera by RT-PCR. Then the phylogenetic tree was constructed by MEGA5.1, and the protein structure was predicted and analyzed by website Psipred and SWISS-MODEL. The DHPR gene of Apis Mellifera was expressed in EsMolecular Cloning, Sequence Analysis and Prokaryotic Expression of DHPR Gene in Apis Mellifera.cherichia coli. The recombinant protein was purified by Ni agarose affinity chromatogrMolecular Cloning, Sequence Analysis and Prokaryotic Expression of DHPR Gene in the European Honeybee, Apis Mellifera (Hymenoptera: Apidae)aphy. Sequence analysis revealed an open reading frame (ORF) of 237 bp, which was named AmDhpr and encodes 237 amino acids. The isoelectric point of the encoded protein is 8.90. Phylogenetic analysis showed that the DHPR proteins of Apis Mellifera, Apis dorsata and Apis florea cluster together. Protein secondary structure prediction showed that AmDHPR contains eight α-helix and eight β-fold regions. By using homology modeling, the predicted three-dimensional structure of AmDHPR was obtained. The AmDhpr ORF was ligated into the expression vector PET-30a and then transformed to E.coli BL21( DE3) for prokaryotic expression and the recombinant protein was purified. Finally, the recombinant DHPR protein with a size of 25 KD was obtained, which was consistent with the expected size. This study successfully cloned the DHPR gene AmDhpr from Apis Mellifera, analyzed its sequences and obtained the purified recombinant protein, providing referencs and materials for further study of the functions of the gene.
Keywords: Apis Mellifera dihydropterin reductase tetrahydrobiopterin sequence analysis structure prediction prokaryotic expression
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