猪源大肠杆菌耐热肠毒素基因的分子克隆

MOLECULAR CLONING OF AN ESHERICHIA COLI HEATSTABLE ENTEROTOXIN GENE FROM PORCINE ORIGIN

以ＰＣＲ方法克隆得到猪源大肠杆菌ＳＴ１基因缺失ＣｏｏＨ端最后两个编码密码和终止密码的ＤＮＡ片段，并以该片段为探针，筛选了以载体ｐＢｌｕｅｓｃｒｉｐｔ构建的猪大肠杆菌Ｐ５５质粒ＤＮＡ文库，得到插入片段约５．５ｋｂ阳性克隆，亚克隆了约０．５ｋｂ含ＳＴ基因的ＴａｑⅠ酶切片段，并对该基因核苷酸序列进行了分析。应用竞争性ＥＬＩＳＡ测定了ＳＴ基因在不同启动子调控下的表达情况，在Ｔ７启动子调控下。

オhe plasmid encoding Esherichia coli heatstable enterotoxin(ST) was isolated from local porcine strain P55 and the ST DNA regions of the 5.5kb Pst Ⅰ fragment were cloned into pBluescript with using the ST probe amplified by PCR.The 0.5kb Taq Ⅰ fragment containing ST gene was finally subcloned and its nucleotide sequence was determined.The Cterminal 18 amino acids deduced were corresponded to the sequence of the ST Ⅰa peptide.The ST gene was hyperexpressed using the phage T7 promoter.The yield of ST was determined with a competitive ELISA.