c血清型变形链球菌致龋相关基因/DNA片段的批量克隆与高通量筛选

目的筛选含致龋相关基因／DNA片段的阳性克隆，为探究变形链球茵致龋的分子机制奠定基础。方法将抑制消减杂交方法获得的高毒力株特异的消减PCR产物与T／A栽体pCR2．1连接，将连接产物转化E．coli TOP0F’感受态细胞，进行蓝白筛选。对96个转化子进行Colony PCR，将PCR产物点样于同一尼龙膜上，分别与地高辛标记的变形链球茵高、低毒力株基因组DNA／AluI酶切产物杂交，高通量筛选阳性克隆。结果随机挑取了96个白色或白色中央有蓝色的茵落，经过Colony PCR，其中有1个转化子的扩增产物为双带，其余均为0．2～2kb之间的单一条带。经过杂交，以与高毒力株基因组有杂交信号，而与低毒力株基因组无杂交信号或信号明显弱的转化子为阳性克隆。筛选的阳性率为50％左右，阳性克隆中含有c血清型变形链球茵致龋相关的基因／片段。结论对抑制消减杂交方法获得的变形链球茵高毒力株特异的消减PCR产物进行批量克隆和高通量筛选，获得了致龋相关的基因／DNA片段。

Batch cloning and a high throughput screening for virulent genes of Streptococcus mutans

Objective To screen the cariogenic genes or DNA fragments and to provide some explanations for the cariogenesis of Streptococcus mutans.Methods The subtractive PCR products between virulent and avirulent strain of Streptococcus mutans by suppression subtractive hybridization were inserted into pCR2.1, a T/A cloning vector. The ligated DNAs were transformed into E.coli TOP10F'competent cells and incubated for proper blue/white color development. The inserts of 96 transformants were colony PCR amplified and arrayed on nylon membrane. After genomic DNA/AluI digest of virulent and avirulent strain were labeled with digoxigenin, the nylon membranes were then hybridized respectively with the two DNA probes for a high throughput screening for positive clones.Results Ninety-six white or white with blue centers colonies were randomly picked. The PCR product from one of the bacterial colonies contained two inserts and others contained single insert whose size ranged from 0.2kb to 2kb. After the differential screening, clones, which hybridized to the DNA probe of virulent strain but not to the probe of avirulent strain, or hybridized to the latter but the signal intensity was much fainter than the former, were chosen as positive clones whose inserts were candidates for the virulent genes or DNA fragments of Streptococcus mutans. About 50% of the clones were confirmed as positive clones.Conclusion The virulent strain-specific subtractive products obtained by suppression subtractive hybridization were cloned. After a high throughput screening, the cariogenic-related genes or DNA fragments were obtaind.