c血清型变形链球菌抑龋相关基因文库的构建

Construction of a suppressive gene library of serotype c Streptococcus mutans

目的: 构建c血清型变形链球菌抑龋相关基因的抑制消减文库,为变形链球菌抑龋基因的筛选奠定基础.方法:从一对c血清型变形链球菌高、低毒力株中提取基因组DNA,AluⅠ酶切,以低毒力株的DNA酶切产物为tester DNA,高毒力株的DNA酶切产物为driver DNA,tester DNA连接接头后与driver DNA进行抑制消减杂交,并检测连接效率与消减效率.将获得的消减PCR产物,即差异基因/差异DNA片段,与pCR2.1载体连接,转化E.coli TOP10F′感受态细胞,进行蓝白筛选.结果:AluⅠ酶切高、低毒力株基因组DNA,产生的酶切产物位于0.1～2 kb之间.tester DNA与接头的连接效率＞25%,抑制消减杂交后消减效率检测显示,同时存在于tester与driver DNA中的23S rRNA基因在消减组中出现时间较未消减组晚12 个循环,将消减PCR产物克隆后,挑取96个转化子,构建抑龋相关基因的抑制消减文库.结论:利用抑制消减杂交技术进行c血清型变形链球菌高、低毒力株之间基因组DNA的比较,初次构建了抑龋相关基因的抑制消减文库.

Objective:To construct a suppression subtractive library of suppression-related genes from c serotype Streptococcus mutans (S.mutans). Methods:After being isolated from virulent and avirulent strain of S. mutans respectively, the intact and high-pure genomic DNAs were digested with restriction enzyme AluⅠ. The digested DNA of the avirulent strain ligated with an adaptor was used as tester DNA, and that of the virulent strain as driver DNA. Then the suppression subtractive hybridization(SSH) was carried out, the efficiency of ligation and subtraction were detected respectively. The subtracted fragments were inserted into vector pCR2.1 using T/A cloning kit and transformed into E. coli TOP10F' competent cells. The white colonies were selected to construct the suppression subtractive library. Results: Through electrophoresis of AluⅠ-digested DNAs, a smear ranged from 0.1 to 2.0 kb was observed. The ligation efficiency of tester DNA with adaptor was at least higher than 25 percent. The subtraction efficiency confirmed the success in enrichment of differential genes between virulent and avirulent strain of S. mutans. In the subtracted group, the appearance time of the 23S rRNA gene in both tester and driver DNA was later than that in the unsubtracted group by twelve cycles. It suggested that suppression subtractive hybridization happened indeed. Then the subtracted fragments were cloned and the suppression-related gene library between virulent and avirulent strain of S. mutans was constructed. Conclusions:The suppression subtracted library of suppression-related genes has been constructed.