大肠杆菌耐热性肠毒素ST_1/K99/LacZ基因重组的研究

Study on Gene Recombination of E coli Heat stable Enterotoxin Gene with K99 Gene and LacZ Gene

将构建的ｐＢＳＴ２～６工程菌质粒用限制性内切酶ＢａｍＨＩ／ＢｇｌⅡ酶切，经琼脂糖凝胶电泳和电洗脱，回收１４７ｂｐ的目的ＳＴ１基因。随之将该基因分别重组到能有效表达Ｋ９９菌毛抗原和ＬａｃＺ酶的ｐＧＫ９９之Ｋ９９基因ＢｇｌⅡ位点和ｐＵＣ１８的ＢａｍＨＩ位点中。通过ＳＴ１基因探针菌落原位杂交、特定酶切分析及ＤＮＡ序列分析，筛选并鉴定出了理想重组子，从而构建出了能分别表达ＳＴ１融合基因产物的工程菌株ｐＳＫ２１９和ｐＸＳＴ１。

The ST1gene of ETEC was amplified by PCR from agene engineering stra in pSLM004.The ST1gene fragment was inserted into the HincⅡsite of vector pUC18by blunt end ligation.The transformants were screened by clony hybridization in situ with α 32 PdATP labelling ST1gene probe,and identified by restriction endonuclease analysis and DNA sequencing.The recom binant plasmid pBST2 6was constructed,which contained ST1gene with BglⅡand Bam HI site (150bp).ST1gene fragments(147bp)obtained from pBST2 6by BglⅡand Bam HI digestion were re spectively inserted into the BglⅡsite of K99gene in pGK99,which could effectively express K99fim briae,and the Bam HI site of polycloning site of pUC18.In this way two kinds of recombinant plas mids,pSK219carrying ST1 K99fusion gene and pXST1carrying ST1 LacZ fusion gene,were suc cessfully constructed.The recombinant strains,pSK219and pXST1,were demonstrated to express ST1 K99fusion protein fimbriae and ST1 LacZ enzymatic fusion protein respectively.