大劣按蚊TEP1 cDNA的克隆和生物信息学分析  

Cloning and bioinformatic analysis of cDNA fragment of TEP1 gene of Anopheles dirus

作　　者：胡晓飞[1,2] 王英[2] 潘春江[1] 刘涛[1] 谢佳[3] 何谐[2] 姜晓梅[4] 黄复生[2] 

机构地区：[1]第三军医大学学员旅7队,重庆400038 [2]第三军医大学基础医学部病原生物学教研室,重庆400038 [3]第三军医大学学员旅20队,重庆400038 [4]第三军医大学学员旅21队,重庆400038

出　　处：《第三军医大学学报》2009年第11期993-996,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30800959);第三军医大学基础部学员创新基金(JCB007)~~

摘　　要：目的克隆大劣按蚊(Anopheles dirus)含硫酯键蛋白1(thioester-containing protein 1,TEP1)的cDNA,并分析其序列。方法根据已报道的冈比亚按蚊等多种昆虫的TEP1氨基酸保守序列设计简并引物,以大劣按蚊血淋巴细胞总RNA为模板,进行RT-PCR扩增。对目的片段纯化回收,经TA克隆后测定其核苷酸序列。利用DNASIS程序分析该序列,并推导其氨基酸序列,最后利用BLAST和DNASIS等进行不同昆虫TEP1氨基酸序列的比对。结果RT-PCR扩增出一约770 bp的条带,TA克隆后DNA测序发现,阳性克隆的核苷酸序列长774 bp,推导其氨基酸序列长258 aa。生物信息学分析表明,该序列与冈比亚按蚊、阿拉伯按蚊、斯氏按蚊TEP1基因的氨基酸序列同源性分别达72%、72%和59%,与冈比亚按蚊的TEP4氨基酸序列同源性为59%。结论从大劣按蚊血淋巴细胞中成功克隆出了TEP1的cDNA部分序列。Objective To clone the cDNA fragment of thioester-containing protein 1 （TEP1） of Anopheles dirus and analyze the sequence with bioinformatic methods. Methods The target cDNA fragment was amplified by RT-PCR with degenerated primers designated based on the conserved amino acid sequences of TEP1 of Anopheles gambiae and other insects. And total RNA from haemocytes of Anopheles dirus was used as the template of RT-PCR. The PCR product was purified and T/A cloned, followed by sequencing the positive clone. Then the sequence was analyzed with DNASIS software and the amino acid sequence was deduced. The obtained amino acid sequence was aligned with TEP1 amino acid sequences of the other insects with BLAST and DNASIS. Results A band of approximately 770 bp was amplified by RT-PCR. A nucleotide acid sequence with 774 bp in length was obtained by T/A cloning and sequencing. The deduced amino acid sequence was 258 aa in length, and had a sequence homology of 72% , 72% and 59% respectively when compared with the TEP1 sequences of Anopheles gambiae, Anopheles arabiensis and Anopheles stephensi. Conclusion The cDNA fragment of TEP1 is successfully cloned from the haemocytes of Anopheles dirus, which provides a foundation for further study on function of the molecular.

关 键 词：大劣按蚊 TEP1 RT—PCR 生物信息学 天然免疫 

分 类 号：R318.04[医药卫生—生物医学工程] R384.1[医药卫生—基础医学]