结核分支杆菌基因文库的构建

目的建立结核分支杆菌基因文库，为其基因及抗原的鉴定分析进而评估它们在诊断、预防、耐药及致病性等方面的作用提供有效手段。方法结核分支杆菌Ｈ３７Ｒａ染色体ＤＮＡ经脱氧核糖核酸酶Ⅰ（ＤＮａｓｅⅠ）部分消化后从凝胶中回收４～８ｋｂ片段，两端接以ＥｃｏＲⅠ接头，并将其连接在λｇｔ１１臂，用噬菌体包装蛋白“包装”，再进行连续稀释，然后将不同稀释度的噬菌体转染宿主菌Ｙ１０９０。结果基因库效率为８５％，滴度为３×１０５ｐｆｕ／ｍｌ，共含１．３×１０５个重组体，平均插入片段长度为３．５ｋｂ。结论该基因库能提供足够的克隆以覆盖Ｈ３７Ｒａ基因。

Construction of mycobacterium tuberculosis genomic library

OBJECTIVE: To provide effective tools for identification and characterization of M. tuberculosis genes/antigenes and to evaluate their roles in diagnosis, vaccination, drug resistance, and pathogenesis. METHODS: M. tuberculosis genomic DNA obtained from H 37 Ra strain was partially digested with DNase I. The DNA fragments ranging from 4-8 kb were isolated from agarose gel and ligated to EcoR I adaptor, and the products were linked to lamda gt11 arms and packaged using an in vitro packaging extract. The different diluted bacteriophages were used to transfect bacteria Y1090. RESULTS: The efficiency and titer of the library were 85% and 3 x 10(5) pfu/ml, respectively. The library contained 1.3 x 10(5) individual recombinant phage whose foreign DNA inserted fragment size was 3.5 kb on average. CONCLUSION: The genomic DNA library constructed here can provide sufficient clone to cover H37Ra gene.