家蝇伴侣蛋白TCP-1基因的序列分析、克隆及诱导表达

生物技术通报 ›› 2014, Vol. 0 ›› Issue (10): 151-155.

家蝇伴侣蛋白TCP-1基因的序列分析、克隆及诱导表达

赵学军, 国果, 吴沁怡, 陶如玉, 吴建伟

贵阳医学院基础医学院,贵阳 550004

收稿日期:2014-02-20 出版日期:2014-10-20 发布日期:2014-10-17
作者简介:赵学军,男,硕士研究生,研究方向：医学寄生虫的分子免疫
基金资助:
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）

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

Zhao Xuejun, Guo Guo, Wu Qinyi, Tao Ruyu, Wu Jianwei

School of Basic Medical Sciences,Guiyang Medical College,Guiyang 550004

Received:2014-02-20 Published:2014-10-20 Online:2014-10-17

摘要/Abstract

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-TCP Ⅰ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-TCP Ⅰ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-TCP Ⅰ重组表达质粒,重组蛋白在大肠杆菌BL21（DE3）中诱导表达。

关键词: 家蝇, TCP, 序列分析, 基因克隆, 表达

Abstract: 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-TCPⅠ））, 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 pI of 5.92, which acid sequence as tcp-1 belong to HSP60 family. The gene coding for MD-TCP Ⅰwas 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）.

Key words: Musca domestic, TCP, Sequence analysis, Gene cloning, Express

赵学军, 国果, 吴沁怡, 陶如玉, 吴建伟. 家蝇伴侣蛋白TCP-1基因的序列分析、克隆及诱导表达[J]. 生物技术通报, 2014, 0(10): 151-155.

Zhao Xuejun, Guo Guo, Wu Qinyi, Tao Ruyu, Wu Jianwei. Sequence Analysis,Cloning and Induced Expression of Chaperonin Gene in Housefly（Musca domesitca）[J]. Biotechnology Bulletin, 2014, 0(10): 151-155.

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