化脓性链球菌高毒力株特异基因文库的构建和分析

目的构建化脓性链球菌高毒力株特异基因文库,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技术(SSH),以从患者体内所分离链球菌为毒力菌株,分离特异表达基因,将其与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,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消减文库后,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100～2 000bp,连接效率大于50%,消减组与非消减组差异明显,成功构建了化脓性链球菌毒力株特异基因消减文库,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SSH及T/A克隆技术成功构建了人源化脓性链球菌高毒力株基因文库,5个未知的新序列有待于进一步的研究。

Construction and analysis of virulence genes subtracted library of streptococcus pyogenes by suppression subtractive hybridization

Objective To construct the virulence genes subtracted library of streptococcus pyogenes and to lay the foundations for screening the virulent genes.Methods Suppression subtractive hybridization was adopted 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 and amplication of the library was carried out with transformation of E.coil TOP10.Dot blot was used to screen the subtracted library,the differentially expressed cDNA fragments were 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 percent.The difference between subtractive group and unsbtractive group was apparently significant.Partial positive clones in the library were randomly selected and successfully sequenced.5/12 sequence showed no homology and presumably represent unknown genes,7/12 had a high similarity to the known genes.Conclusion The virulence genes subtracted library of streptococcus pyogenes is constructed successfully with SSH and T/A cloning techniques.The library is efficient and lays solid foundation for screening and cloning new and specific virulence genes of streptococcus pyogenes.