文档介绍：
大肠杆菌 W3110 菌株 waal 基因的敲除及糖
基转移酶 pglB 的表达#
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(1. 山东大学药学院;
2. 山东大学国家糖工程技术研究中心,济南 250012)
摘要:目的:获得敲除糖基转移酶 waal 基因的大肠杆菌,并在 waal 基因敲除的大肠杆菌中
表达来源于空肠弯曲菌的另外一种糖基转移酶 pglB。方法:PCR 扩增获得两端含有 waal 同
源基因的 PCR 同源重组片段,把该 PCR 同源重组片段导入到含有 Red 基因重组表达质粒 pKD46
的 w3110 中,获得 PCR 同源重组片段置换 waal 基因的克隆菌株。然后把温度敏感
型质粒 pCP20 导入该克隆菌株,最终获得 waal 基因、PCR 同源重组片段和质粒 pCP20 均消
除的克隆菌株 w3110/△waal;并且通过转化质粒 pBAD/Myc-His/pglB,优化表达条
件,在 w3110/△waal 中表达 pglB 蛋白。结果:获得了 waal 基因敲除的 w3110,
并且在 w3110 中表达获得了 pglB 蛋白。
关键词: 微生物与生化药学;waal 基因;pglB 基因;大肠杆菌 w3110
中图分类号:Q812
Deletion of waal gene in W3110 and expression of
pglB
ZHANG Xiaobing1,2, HAN Xiqian1,2, SHI Yikang1,2
(1. School of Pharmaceutical Sciences,Shandong University;
2. National Glycoengineering Research Center,Shandong University,Jinan 250012)
Abstract: Object: To obtain a strain of W3110 deleting waal gene and express pglB protein
in W3110 without waal gene. Methods: Red bination system from bacteriophage λ
was used for genome deletion. PCR products for homogenous bination were obtained by
using primers containing oligonucleotides which were homologous to sequences upstream and
downstream of waal gene. PCR binant products were then introduced by electroporation
into E coli W3110 which contain plasmid pKD46 expressing Red bination proteins. Wa