Construction of a suppressive gene library of serotype c Streptococcus mutans / 实用口腔医学杂志

ABSTRACT

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.