大肠杆菌耐热性肠毒素ST_1－LacZ融合基因的构建

Construction of Fusion Gene for Enterotoxigenic Es-cherichia coli Heat-Stable Enterotoxin I and LacZ GeneEncoding β-Galactosidase

利用含大肠杆菌耐热性肠毒素ＳＴ１基因的质粒ｐＢＳＴ２－６和含ＬａｃＺ基因（编码β－半乳糖苷酶）的载体ｐＵＣ１８，构建成ＳＴ１－ＬａｃＺ融合基因。将质粒ｐＢＳＴ２－６用限制性内切酶ＢａｍＨＩ和ＢｇｌⅡ消化，经３％琼脂糖凝胶电泳分离、透析袋电洗脱法回收１４７ｂｐＤＮＡ片段，再将载体ｐＵＣ１８用ＢａｍＨＩ消化、碱性磷酸酶处理，最后将处理好的ｐＵＣ１８ＤＮＡ与１４７ｂｐＳＴ１ＤＮＡ通过Ｔ４ＤＮＡ连接酶进行粘性末端连接，转化至受体菌ＤＨ５ａ中。通过菌落原位杂交筛选，共筛选出５３个为ＳＴ１基因探针杂交阳性的菌落。菌落原位杂交阳性的菌株经培养和抽提纯化质粒后，经限制性内切酶（ＥｃｏＲＩ／ＸｂａＩ和ＥｃｏＲＩ／ＢａｍＨＩ）酶切分析和ＤＮＡ序列分析，证明重组质粒ｐＸＳＴ１含有ＳＴ１基因，而且ＳＴ１基因在重组ＤＮＡ中连接方向是正确的，具有正确的阅读框架。再者，ＤＨ５ａ（ｐＸＳＴ１）菌株能在含ｘ－Ｇａｌ的ＬＢ平板上长成蓝色菌落，表明该菌株能表达具有β－半乳糖苷酶活性的大肠杆菌耐热性肠毒素ＳＴ１－β－半乳糖苷酶融合蛋白。ＳＴ１－ＬａｃＺ融合基因的构建，为研究ＳＴ的免疫原性和抗ＳＴ抗体在预防产肠毒素性大肠杆菌（ＥＴＥＣ）感染中的作用?

Using recombinant plasmid pBST2-6 containing enteriotoxigenicEscherichia coli heat-stable enterotoxin I(ST1) gene and vector pUC18 containingLacZ gene(encoding β-galactosidase),we constructed fusion gene ST1-LacZ.We usedthe restriction endonucleases Bam HI and Bgl-Ⅱ to cleave the 147 bp ST1 gene fragmentfrom plasmid pBST2-6,isolated by 3% agarose gel electrophoresis, recovered the 147bp ST1 DNA fragment,then we inserted it into an expression vector pUC18 whichcleaved with Bam HI and treated with CIAP, by bluntend ligation. The transformantswere screened by ST1gene probe that was labeled with α-32P dATP by PCR gene ampli-fying-labeling method.The recombinant plasmid pXST1 was studied in detail by restric-tion endonuclease analysis and DNA sequencing. The results have shown that the re-combinant plasmid carried ST1 and LacZ gene fragments and had positive reading frame.By transformation of E.coli strain DH5a, we got DH5α (pXST1).The strain can pro-duce two kinds of antigen which we proved by using SDS-PAGE,and can be used ascandidate of live vaccine strain and establishment of McAb-ELISA.