LSPA1早期基因gp22细菌双杂交诱饵载体与筛选文库的建立

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.033

生物技术通报 ›› 2015, Vol. 31 ›› Issue (7): 214-219.doi: 10.13560/j.cnki.biotech.bull.1985.2015.07.033

LSPA1早期基因gp22细菌双杂交诱饵载体与筛选文库的建立

冯梦蝶¹, 毛普加¹, 洪愉², 许泽仰¹, 赵继华²,, 杨洪文² 宋武战², 黄芬¹, 井申荣¹, 曾韦锟^1，3

(1.昆明理工大学医学院，昆明 650500；2.成都军区昆明总医院核医学科，昆明 650032；3.昆明学院医学院临床检验教研室，昆明 650214)

收稿日期:2014-11-03 出版日期:2015-07-16 发布日期:2015-07-16
作者简介:冯梦蝶，女，硕士研究生，研究方向：病原微生物；E-mail：fengmengdier@163.com
基金资助:
国家自然科学基金项目(31160193)，云南省教育厅科学研究基金项目(2010Y398)，云南省应用基础研究面上项目(2010ZC055；2012FB135)

Construction of LSPA1 Early Gene gp22 Bacterial Two-hybrid Bait Vector and Screening Library

Feng Mengdie¹, Mao Pujia¹, Hong Yu², Xu Zeyang¹, Zhao Jihua², Yang Hongwen², Song Wuzhan², Huang Fen¹, Jing Shenrong¹, Zeng Weikun^1，3

(1. Medical Faculty，Kunming University of Science and Technology，Kunming 650500；2. Department of Nuclear Medicine，Kunming General Hospital of Chengdu Military Command，Kunming 650032；3. Department of Clinical Laboratory of Medical Faculty，Kunming University，Kunming 650214)

Received:2014-11-03 Published:2015-07-16 Online:2015-07-16

摘要/Abstract

摘要：

构建细菌双杂交系统中的诱饵载体pKT25-gp22和甲型副伤寒沙门氏菌基因文库以便后续的筛选实验。PCR扩增获得gp22基因，插入pKT25构成诱饵质粒pKT25-gp22。提取甲型副伤寒沙门氏菌基因组DNA，经Sau3AⅠ部分酶切后连接到pUT18C质粒的BamHⅠ位点，获得基因组DNA表达文库。用化转的方法将诱饵质粒与pUT18C共转入BTH101，检测诱饵质粒自激活作用，并检测诱饵蛋白对宿主菌的毒性。将文库质粒电转至JM109感受态，PCR鉴定文库的多样性。诱饵载体pKT25-gp22无自激活报告基因的能力且对细菌的生长无毒性。所构建的副甲基因组文库覆盖基因组达8倍，满足文库筛选需要。成功构建细菌双杂交系统中诱饵载体pKT25-gp22，筛选文库质量良好，可应用于细菌双杂交实验。

关键词: 细菌双杂交, 诱饵载体, 基因组文库, 早期基因, 甲型副伤寒沙门氏菌

Abstract:

This study aims to construct a bait vector pKT25-gp22 and the gene library of Salmonella paratyphi A in bacterial two-hybrid system for further screening study. The gp22 gene was obtained by PCR amplification and then cloned into pKT25 to constitute the bait plasmid pKT25-gp22. The genomic DNA of S. paratyphi A was extracted and partially digested with Sau3AⅠ. The fragments between 250 to1 500 bp were re-natured and ligated to BamHⅠsite of pUT18C plasmid, and the genomic DNA library was obtained. The bait plasmid pKT25-gp22 and pUT18C were co-transformed to BTH101, and the self-activation of bait plasmid pKT25-gp22 and the toxicity of bait protein to the host bacteria were detected. The library plasmids were transformed to JM109 competence and the library diversity was identified by PCR. The results revealed that the bait vector was successfully constructed without self-activation of the transcription of reporter gene and non-toxic to the growth of bacteria. The genomic library covering 8 times of S. paratyphi A genome met the requirements of screening. In conclusion, the bait plasmid pKT25-gp22 and the genomic DNA library of S. paratyphi A were successfully constructed, which can be utilized in bacterial two-hybrid study.

Key words: bacterial two-hybrid system, bait vector, genomic library, early gene , Salmonella paratyphi A

冯梦蝶, 毛普加, 洪愉, 许泽仰, 赵继华, 杨洪文宋武战, 黄芬, 井申荣, 曾韦锟. LSPA1早期基因gp22细菌双杂交诱饵载体与筛选文库的建立[J]. 生物技术通报, 2015, 31(7): 214-219.

Feng Mengdie, Mao Pujia, Hong Yu, Xu Zeyang, Zhao Jihua, Yang Hongwen, Song Wuzhan, Huang Fen, Jing Shenrong, Zeng Weikun. Construction of LSPA1 Early Gene gp22 Bacterial Two-hybrid Bait Vector and Screening Library[J]. Biotechnology Bulletin, 2015, 31(7): 214-219.

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LSPA1早期基因gp22细菌双杂交诱饵载体与筛选文库的建立

Construction of LSPA1 Early Gene gp22 Bacterial Two-hybrid Bait Vector and Screening Library

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