利用反向PCR方法扩增细菌热激蛋白HSP60基因

Amplification of Bacterial Heat Shock Protein 60 Gene Using Inverse PCR Method

利用PCR简并引物扩增出HSP6 0基因中一段约 6 0 0bp的核心片段 ,将该核心片段标记为探针 ,与基因组DNA进行Southern杂交 ,选择出适宜的限制性内切酶 ,以便消化基因组DNA得到大小合适的、含有HSP6 0基因的酶切片段。将酶切片段自身环化后作为模板进行反向PCR ,引物的延伸方向自核心片段出发延环化分子向未知序列区进行 ,可扩增出核心区上下游的序列。应用该方法 ,扩增并测定了寓齿双歧杆菌 (Bifidobacteriumdenticolens)DSM1 0 1 0 5 T、奇异双歧杆菌 (Bifidobacteriuminopinatum)DSM1 0 1 0 7T 和阴道加德纳氏菌 (Gard nerellavaginalis)ATCC1 40 1 8T 的HSP6 0全基因序列及青春双歧杆菌 (Bifidobacteriumadolescentis)JCM1 2 75 T98%以上的HSP6 0全基因序列。结果表明 ,反向PCR方法可有效的扩增细菌HSP6

A method is presented for rapid in vitro amplification of DNA sequences of bacterial HSP60 gene.In our previous work,using degenerate oligonucleotide primers for conserved regions of HSP60 gene,a 600bp fragment was amplified by PCR,cloned and sequenced.An inverse PCR method,with the primers oriented in the reversed direction of the usual orientation,is used to amplify the DNA sequences that flank the 600bp known region.The feasibility of this method is shown by amplifying the complete HSP60 gene sequences of 4 bacterial strains,namely Bifidobacterium denticolens,Bifidobacterium inopinatum,Bifidobacterium adolescentis and Gardnerella vaginalis.