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

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

Biotechnology Bulletin ›› 2015, Vol. 31 ›› Issue (7): 214-219.doi: 10.13560/j.cnki.biotech.bull.1985.2015.07.033

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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 Online:2015-07-16 Published:2015-07-16

Abstract

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

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

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