家蝇伴侣蛋白TCP-1基因的序列分析、克隆及诱导表达  

Sequence Analysis,Cloning and Induced Expression of Chaperonin Gene in Housefly(Musca domesitca)

作　　者：赵学军[1] 国果[1] 吴沁怡[1] 陶如玉 吴建伟[1] 

机构地区：[1]贵阳医学院基础医学院,贵阳550004

出　　处：《生物技术通报》2014年第10期151-155,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(81160204;81360254);贵州省科技厅基金项目(黔科合[2010]3160);高校博士点学科专项科研基金项目(20105215120001)

摘　　要：旨在对EST筛选得到的家蝇伴侣蛋白TCP-1(MD-TCPⅠ)基因进行序列分析,克隆其cDNA序列并在大肠杆菌中诱导表达。采用EST测序技术从已构建的家蝇幼虫cDNA质粒文库中筛选到MD-TCPⅠ基因,对其进行序列测定和分析。以该基因的cDNA文库质粒为模板,通过PCR的方法进行扩增,以pET-28a(+)为载体构建重组质粒,再转化到表达宿主大肠杆菌BL21(DE3)中,IPTG诱导表达。表达产物通过SDS-PAGE进行鉴定。结果显示,MD-TCPⅠ基因ORF全长753 bp,编码250个氨基酸,理论分子量27.07 kD;等电点5.92,该序列编码的蛋白属于热休克蛋白60家族的TCP。构建了正确基因序列MD-TCPⅠ重组表达质粒,重组蛋白在大肠杆菌BL21(DE3)中诱导表达。The aim of this study is to analyze and predict the structural and characteristics of genes and encoding proteins of MD-TCP I （ Musca domesitca Chaperonin TCP- 1 （ MD-TCP I ） ） , with the methods of cloning and expressing that gene. Sequence analysis indicated that the open reading frame was 753 bp, encoding a putative protein consisting of 250-amino acids, which no signal sequence and NCBI-BLAST showed acid sequence identify with other insect TCP-1 were 89%. The protein, with a predicted molecular weight of 27.07 kD, and p[ of 5.92, which acid sequence as tcp-1 belong to HSP60 family. The gene coding for MD-TCP I was amplified by polymerase chain reaction （ PCR ） , and then was ligated into vector pET-28a （ ＋ ） and transformed into Escherichia coli BL21 （ DE3 ） competent cell, induced with IPTG. The fusion protein in the expression vector was analyzed by SDA-PAGE. The results indicated that the recombinant plasmid with the correct target gene was constructed, and the fusion protein was expressed in E. coli BL21 （ DE3 ） .

关 键 词：家蝇TCP 序列分析 基因克隆 表达 

分 类 号：Q78[生物学—分子生物学]