利用抑制消减杂交技术构建变形链球菌高毒力株特异的基因文库

Objective To construct a suppression subtractive library of virulence-related genes from c serotypeStreptococcus mutans(S.mutans), and lay foundations for screening the virulent genes.Methods After being isolated from virulent and avir- ulent strain ofS.mutansrespectively, the intact and high-pure genomic DNAwas digestedwith three appropriate four-base-cutting restriction endonucleases to produce fragments of optimal length. The digested DNA of the virulent strain ligated with adaptorwas used as tester DNA, and that of the avirulent strain as driver DNA. Then the suppression subtractive hybridizationwas carried out, and the efficiency of ligation and subtraction detected respectively. The subtracted fragmentswere inserted into vector pCR2.1 us- ingT/A cloning kit, and transformed intoE.coliTOP10F′competent cells. Those white colonies were selected to construct the suppression subtractive library.Results AluⅠchosen from three restriction endonucleases was verified to be suitable for prepar- ing restriction fragments fromS.mutansgenomic DNA. Through electrophoresis ofAluⅠ-digested DNA, a smear ranged from0·1 to 2·0 kbwas observed. The ligation efficiency of tester DNAwith adaptorwas at least higherthan 25 percent. The subtraction ef- ficiency of suppression subtractive hybridization confirmed the success in enrichment of differential genes between virulent and avir- ulent strain ofS.mutans. In the subtracted group, the appearance time of the 23S rRNAgene both in tester and driver DNAwas later than that in the unsubtracted group by six cycles. It suggested that suppression subtractive hybridization happened indeed. After the subtracted fragmentswere cloned, 96 colonieswere picked up for constructing the suppression subtractive library of viru- lence-related genes ofS.mutans.Conclusion Suppression subtractive hybridization allows rapid and easy construction of viru- lence-related gene library ofS.mutans.