庆大霉素耐药基因探针的构建

Construction of a Gentamicin Resistance Gene Probe

本文从致痛性大肠杆菌的pEFM2质粒出发,克隆获得重组质粒pBY101,绘制了pBY101的PstⅠ、EcoRⅠ及PvuⅡ的限制性酶切物理图谱,确定Gm耐药基因在8.9kb的pstⅠ—1片段上。用EcoRⅠ酶解pBY101,再连接,次级克隆获得重组质粒pBY102。确证pBY102的4.9kb PstⅠ—EcoRⅠ片段含有编码Gm耐药基因。采用低熔点琉脂糖挖块法回收4.9kb片段,以生物素-7-dAT标记之,并作为探针,以菌落原位杂交珐检测106株Gm耐药性细菌,而且证明本研究构建的Gm—DNA探针与国外引进的ANT(2″)基因探针不同源。

Gentamicin resistance gene carried on plasmid pEFM2 from enteropathogenic Escherichia coli was shotgun cloned directly into E.coli HB101 by uning the vector pBR322. Recombinant plasmid pBY101 was obtained. Restriction pattern analysis of pBY101 revealed that it carried a 8.gkb PstⅠ fragment of pEFM2 on which the gentamicin resistance genc was located. Furthermore, a restriction map of pBY101 was constructed for PstⅠ, EcoR1 and PvuⅡ by simple and double digestion of plasmid. For reduction of the size of the insert, pBY101 was digested with FcoRJ, religated and transformed into E.coli k 802.The gentamicin resistant subelones were screened for plasmids from which a chimeric plasmid pBY102 was obtained Restriction pattern analysis of pBY102 revealed that it carried a 4.9 kb PstI-EcoRI fragment of pBY101 on which the gentamiein resistance gene was located. This fragment was recovered from low-melting-temperature agarase by the slot method and then labeled with biotin-7-dATP by nick translation with a commercial kit. It was used as a probe for detection of 106 strains of gentamicin resistance Enterobaeteriaeae by colony hybridization. Meanwhile, the 4.9 kb probe showed no homologous with the ANT (2') gene probe from abroad.