猪大肠杆菌热敏肠毒素B亚基基因的克隆及高效表达

CLONING AND EXPRESSION OF AN ESHERICHIA COLI HEAT-LABILE ENTEROTOXIN SUBUNIT B GENE OF PORCINE ORIGIN

本文以多聚酶链反应扩增得到的ＬＴＢ基因ＤＮＡ片段为探针，克隆了含猪源大肠杆菌ＬＴＢ基因的７７５ｂｐＨｉｎｄⅢ酶切片段，对插入片段进行了核苷酸序列测定：将含ＬＴＢ基因的ＥｃｏＲＩ－ＨｉｎｄⅢ酶切片段插入表达载体ｐＫＫ２２３－３中得到亚克隆ｐＲＸ－ＬＴＢ，经ＳＤＳ－ＰＡＧＥ电泳，Ｗｅｓｔｅｒｎ－ｂｌｏｔ和ＥＬＩＳＡ分析，ｐＲＸ－ＬＴＢ转入大肠杆菌ＪＭ１０７细胞中，在ＩＰＴＧ诱导下，ＬＴＢ基因得到高效表达，其表达量为亲本菌株的３２倍。

A heat labile enterotoxin(LT)plasmid was isolated from an E.coli of piglet.The HindⅢ 775bp fragment containing the heat labile enterotoxin subunit B(LTB) gene was cloned in pGEM 3zf(+).The nucleotide sequence of the insert fragment was determined.It showed that the recombinant plasmid pGLT17 contained a truncated form of LT gene,in which the LTA gene lacked the NH 2 terminus and the LTB gene was intact.The LTB gene was subcloned in pKK223 3 and hyperexpressed using Tac promoter with deduction by IPTG in E.coli JM107.The immunological assays showed that the LTB production level in recombinant E.coli cells was 32 times higher than the parental strain.