淡色库蚊氨肽酶N基因的克隆及生物信息学分析

目的 克隆淡色库蚊Bt毒素受体氨肽酶N(Aminopeptidase N,APN)基因,并对基因及编码蛋白进行生物信息学分析.方法 提取淡色库蚊RNA,通过RT-PCR扩增氨肽酶N基因的全长cDNA序列,纯化PCR产物连接至pMD19-T载体,阳性重组质粒经PCR鉴定后进行基因测序.生物信息学分析APN基因的序列同源性及编码蛋白的结构特征.结果 该基因cDNA序列全长为1 383 bp(含终止密码子),编码460个氨基酸,与致倦库蚊XM_001862270.1序列的同源性为100%.预测蛋白质分子量为52.9 kDa,等电点为4.98.前21个氨基酸为N-末端的信号肽,存在2个N-糖基化位点( 93NLT95和¹⁶⁹NVS¹⁷¹).具有肽酶家族M1的序列特征,锌结合位点HEXXH和谷氨酸锌化氨肽酶的保守结构GAMEN.结论 成功克隆了淡色库蚊氨肽酶N基因并对APN蛋白进行生物信息学分析,为进一步探讨淡色库蚊APN的功能及Bt毒素作用机制奠定了基础.

Molecular cloning and bioinformatics analysis on aminopeptidase N of Culex pipiens pallens

In order to clone the gene of aminopeptidase N (APN), the receptor of Bt insecticidal crystal protein of Culex pipiens pallens, and analyze the biological information of the coding protein, single mosquito larva RNA was extracted from Cx. pipiens pallens and the APN cDNA sequence was amplified by RT-PCR. The PCR products were cloned into the pMD-19T simple vectors and sequenced. The sequence homology and the structural feature of the coding protein were analyzed by biological information systems. Results showed that the full-length of CpAPN was 1 383 bp that encoded a protein of 460 amino acid residues. The homology with Culex quinquefasciatus (XM_001862270.1) was 100%. The predicted molecular weight and isoelectric point were 52.9 kDa and 6.33, respectively. It contained an N-terminal signal peptide with 21 amino acids, and two N- glycosylaion sites (⁹³NLT⁹⁵and ¹⁶⁹NVS¹⁷¹), showing the typical characteristics of peptidase family M1, and with the zinc-binding motif HEXXH and the conserved GAMAN motif. Aminopeptidase N gene of Culex pipiens pallens was cloned successfully and analyzed by bioinfomatics technique. The results provided experimental evidence for revealing the function of CpAPN and the insecticidal mechanism of Bt toxin.