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

华西口腔医学杂志

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

郭丽宏1,史俊南2,张　莹2

1.北京大学口腔医学院　生物教研室,北京100081;2.第四军医大学口腔医院　牙体病科,陕西　西安710032

收稿日期:2005-12-25 修回日期:2005-12-25 出版日期:2005-12-20 发布日期:2005-12-20
通讯作者: 郭丽宏,Tel:010-62179977-2535
作者简介:郭丽宏(1972-),女,福建人,副教授,博士
基金资助:
国家自然科学基金资助项目(30271417);北京市自然科学基金资助项目(7042035)

Construction of a Virulence-related Gene Library ofStreptococcus mutansby Suppression Subtractive Hybridization　

GUOLi-hong1,SHI Jun-nan2,ZHANG Ying2

1.Dept.of Biology,College of Stomatology,Peking University,Beijing 100081,China; 2.Dept.of Oral Medicine,College ofStomatology,The Fourth Military Medical University,Xi′an710032, China

Received:2005-12-25 Revised:2005-12-25 Online:2005-12-20 Published:2005-12-20
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摘要/Abstract

摘要： 目的　构建c血清型变形链球菌高毒力株特异的基因文库,为变形链球菌毒力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,用四碱基限制性内切酶酶切,以高毒力株的DNA 酶切产物为tester DNA,低毒力株的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产物与pCR2.1载体连接,转化E.coliTOP10F′感受态细胞, 进行蓝白筛选。结果　从识别四碱基序列的限制性内切酶中,筛选出AluⅠ,用于制备变形链球菌基因组DNA的代表性片段,产生的酶切产物位于0·1~2.0 kb。tester DNA与接头的连接效率大于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6个循环,将消减 PCR产物克隆后,挑取96个转化子,构建高毒力株特异的基因文库。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,初次构建了高毒力株特异的基因文库。

关键词: 变形链球菌, 抑制消减杂交, 基因组DNA

Abstract: 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.

Key words: Streptococcus mutans, suppression subtractive hybridization, genomic DNA

郭丽宏;史俊南;张莹. 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[J]. 华西口腔医学杂志.

GUOLi-hong1,SHI Jun-nan2,ZHANG Ying2. Construction of a Virulence-related Gene Library ofStreptococcus mutansby Suppression Subtractive Hybridization　[J]. West China Journal of Stomatology.

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