大肠杆菌不耐热性肠毒素LT_B基因的克隆及其核苷酸序列分析  

CLONING AND NUCLEOTIDE SEQUENCING OF THE HEAT LABILE ENTEROTOXIN B SUBUNIT GENE IN ESCHERICHIA COLI

作　　者：许崇波[1,2,3] 卫广森[1,2,3] 吴广谋[1,2,3] 张立国 冯书章 刘晓明[1,2,3] 

机构地区：[1]解放军农牧大学军事兽医研究所 [2]辽宁省兽医生物药品厂 [3]南京农业大学微生物教研室

出　　处：《畜牧与兽医》1997年第4期154-156,共3页Animal Husbandry & Veterinary Medicine

摘　　要：用内切酶ＳａｃⅠ和ＨｉｎｄⅢ双酶切大肠杆菌质粒ｐＥＷＤ２９９，回收５０５ｂｐ的ＬＴＢ基因片段，再将载体ｐＵＣ１８用ＳａｃⅠ和ＨｉｎｄⅢ双酶切，最后将ｐＵＣ１８ＤＮＡ与５０５ｂｐ的ＬＴＢＤＮＡ进行连接，转化至受体菌ＤＨ５α中，经ＳａｃⅠ／ＨｉｎｄⅢ、ＥｃｏＲⅠ／ＨｉｎｄⅢ酶切反应鉴定重组子，得到了理想重组子质粒ｐＸＬＴ１。再将ｐＸＬＴ１进行ＥｃｏＲⅠ酶切、大肠杆菌聚合酶ⅠＫｌｅｎｏｗ大片段补平、ＨｉｎｄⅢ酶切处理，然后与用ＨｉｃⅡ和ＨｉｎｄⅢ酶切的ｐＵＣ１８连接，转化至受体菌ＤＨ５α中，经ＸｂａⅠ／ＨｉｎｄⅢ酶切反应鉴定重组子，得到了理想重组子质粒ｐＸＬＴ１－１。将质粒ｐＸＬＴ１－１进行核苷酸序列分析。We used the restriction endonucleases SacⅠ and HindⅢ to digest the plasmid pEWD299,isolated by 1.5% agarose gel electrophoresis,recovered the 505 bp LT B gene fragment,then we inserted it into the vector pUC18 which digested with SacⅠ and HindⅢ,by blunt end ligation.The ligation mixture was used to transform Escherichia coli DH5α.The transformants were screened on ampiciline/X Gal/IPTG plates.One recombinant plasmid,pXLT1-1,was identified with restriction endonucleases (SacⅠ,HindⅢ,SacⅠ/HindⅢ and EcoRⅠ/HindⅢ).The nucleotide sequence analysis reveals the DNA sequence of heat labile enterotoxin B subunit gene in Escherichia coli.

关 键 词：大肠杆菌 LTB基因 基因克隆 核苷酸序列分析 

分 类 号：S852.612[农业科学—基础兽医学]