化脓性链球菌高毒力株特异基因文库的构建和分析  

Construction and analysis of virulence genes subtracted library of Streptococcus pyogenes

作　　者：吴剑锋[1] 夏永祥[1] 

机构地区：[1]南京医科大学附属南京第一医院医学检验科,210006

出　　处：《国际检验医学杂志》2011年第15期1701-1703,共3页International Journal of Laboratory Medicine

基　　金：南京市卫生局医学科技发展项目(YKK07047)

摘　　要：目的构建化脓性链球菌毒力基因文库，克隆和筛选化脓性链球菌高毒力株特异表达基因。方法采用抑制消减杂交方法，以从患者体内分离的链球菌作为毒力菌株，分离毒力基因，将其与PMDl8-T载体进行T／A连接构建文库，将连接产物转化感受态大肠杆菌TOP10进行文库扩增后，将转化菌液涂布于LB固体平板，构建化脓性链球菌毒力株特异基因消减文库，用斑点杂交初步筛选后，将获得的阳性克隆进行测序和同源性分析。结果酶切产物为100～2000bp，连接效率大于50％，成功构建了化脓性链球菌毒力基因消减文库，所得阳性克隆经斑点杂交筛选后测序，与Genebank数据库进行同源性比对。5个未知序列可能为新基因，7个与已知基因有高度的同源性。结论用抑制消减杂交技术及T／A克隆技术成功构建了化脓性链球菌毒力基因文库；5个未知的新序列可能与化脓性链球菌的高毒力有关。Objective To construct virulence genes subtracted library of Streptococcus pyogenes and lay foundations for screening the virulent genes. Methods Suppression subtractive hybridization was performed to isolate the fragments of virulence genes in Streptococcus pyogenes. Then these fragments were directly inserted into T/A cloning vector to set up subtractive library. Amplication of the library was carried out with transformation of E. coil TOP10. Dot blot was used to screen the subtracted library. The dif ferentially expressed cDNA fragments was sequenced and analyzed in Genebank with Blast search. Results A smear ranged from 100--2 000 bp was observed. The ligation efficiency of tester DNA with adaptor was at least higher than 50%. The difference be tween subtractive group and unsbtractive group was apparently significant. Partial positive clones in the library were randomly se lected and successfully sequenced. 5 sequences showed no homology and presumably represent unknown genes, 7 sequences had a high similarity to the known genes. Conclusion Virulence genes subtracted library of Streptococcus pyogenes was constructed successfully with SSH and T/A cloning techniques. The library could be efficient and lays solid foundation for screening and cloning new and specific virulence genes of Streptococcus pyogenes.

关 键 词：链球菌科 抑制消减杂交 毒力基因 基因消减文库 

分 类 号：R346[医药卫生—基础医学]