卡介苗中具启动子活性片段克隆及分析

CLONING AND CHARACTERIZATION OF PROMOTER FUNCTIONING FRAGMENTS OF BCG

利用启动子探针型质粒ｐＫＫ２３２－８从卡介苗染色体ＤＡＮ的ＢａｍＨＩ酶切片段中克隆到４个具启动功能的ＤＮＡ片段，测定各重组子对氯霉素（Ｃｍ）的抗性，其中含３．３Ｋｂ外源片段的重组质粒ｐＢＣＧ２大肠杆菌转化子在ＬＭ培养基上对Ｃｍ抗性可达１７０×１０－６ｇ／ｍＬ，作了该片段的限制性酶切图谱．对ｐＢＣＧ２利用ＳａｌⅠ位点，亚克隆出４种部分重叠的具启动功能的ＤＮＡ片段，这４种重组质粒分别命名为ｐＢＣＧ２－１，ｐＢＣＧ２－２，ｐＢＣＧ２－６和ｐＢＣＧ２－８，并分析了其中插入片段的大小及其转化子对Ｃｍ的抗性．将ｐＢＣＧ２－２的ＳａｌⅠ外源片段插入到分枝杆菌启动子探针型穿梭质粒ｐＥＱ３，获得一个可在卡介苗中表达ｌａｃＺ基因的重组子ｐＥＢ２２．斑点杂交实验证明，具有启动功能的ＤＮＡ片段来源于卡介苗的染色体ＤＮＡ．

Four DNA fragments,which had the promoter function,were cloned from the Bam HI fragments of BCG (Bacille calmette Guerin) chromosome DNA using the promoter probe vector pKK232 8.The resistance to chloramphenicol(Cm) of the recombinants were tested,and the physical map of the recombinant whose level of Cm resistance got to 170×10 -6 g/mL,named pBCG2 was determined.The insert of pBCG2 was subcloned into pKK232 8 using Sal I,and four Cm resistant recombinant were gotten,named pBCG2 1,pBCG2 2,pBCG2 6 and pBCG2 8.The fragment lengths and Cm resistance of each subclone were determined.The insert of pBCG2 2 was isolated and inserted into promoter probe shuttle vector pEQ3.A recombinant,named pEB22,which could express lac Z gene in BCG,was gotten.Through dot blotting it was confirmed that all the promoter functioning DNA fragments came from BCG chromosome DNA.The result show that a DNA fragment with the promoter activity in BCG and E.coli was cloned.