带有sop基因稳定表达载体的构建

Construction of a Stable Expression Vector Carrying sop Genes

F质粒的第五个EcoRⅠ片段mini-F具有质粒的分配功能。其EcoRⅠ-BamHⅠ片段含有oriS、ccd、repD和sop基因(sopA,B和C)。该片段与pBR322重组,得到质粒pDMC32。pDMC32经SmaⅠ酶切,T4连接酶连接,得到衍生质粒pDMC311,消除了oriS、ccd和repD片段。MI32(pDMC32)和MI311(pDMC311),在液体限磷基础培养基中培养100代,质粒保持率分别为93％和100％。而对照MIR322(pBR322)培养55代时,质粒保持率仅为10％。带有色氨酸启动子质粒pDR720与pBR322重组,得到质粒pDMC40。pDMC40再与mini-F的sop基因重组,得到带有sop基因的稳定表达质粒pDMC48。MI48(pDMC48)在液体限磷基础培养基中培养100代,质粒保持率为100％。

The EcoR I -BamH I fragment of mini-F contains oriS, ccd, rcpD and sop genes (sopA, sopB and sopC). Recombinant plasmid pDMC32 was constructed from EcoR I -BamH I fragments of plasmid pBR322 and mini-F carried by plasmid pFDW29. The plasmid pDMC32 was treated with restriction enzyme Sma I , eliminating oriS,ccdB and repD, the larger fragment ligated itself with T4 DNA ligase. The resulting plasmid pDMC311 contains sop genes and ccd A gene. The E. coli strains MI32 harbouring plasmid pDMC32 and MI311 harbouring plasmid pDMC311 were cultivated in nonselective phosphate-limited basal medium for 100 generations respectively. The maintenance of the plasmids pDMC32 and pDMC311 were 93% and 100% respectively. As for the control plasmid pBR322, only 10% of plasmid maintenance was observed after 55 generations cultivation. In order to make a stable expression vector that carries sop genes,plasmid pDMC40 was constructed by adding a trp promoter from pDR720 to pBR322. The stable expression vector pDMC48 was then derived from pDMC40 by inserting sop genes into it from pDMC311. The maintenance proportion of plasmid pDMC48 in E. coli was still 100% after 100 generations continuous cultivation of cells harbouring the plasmid in phosphate-limited basal medium.