构建链霉菌小型基因文库的新型正选择克隆载体

Positive selection vector for the preparation of Streptomyces genomic mini-library

目的构建链霉菌小型基因文库是获得链霉菌基因的一个重要手段,常规使用的克隆载体常有着一定程度的假阳性,因此构建的高效率的克隆载体有着实际的意义。方法本研究通过重叠延伸PCR手段将一个96bp的多克隆位点序列插入至rpsL基因的启动子和开放阅读框之间,将此DNA片段克隆至pBluescript KS（-）而获得了新型的正选择克隆载体pLS975。结果 pLS975有18个酶切位点可用于外源DNA片段的克隆,重组克隆以氨苄青霉素抗性和链霉素抗性进行筛选,宿主菌为链霉素抗性菌株如Escherichia coli DH10B。两个小型基因文库的制备显示pLS975具有很高的克隆效率（96%~100%）。结论 pLS975可成为构建链霉菌小型基因文库的良好载体。

Objective Preparation of a Streptomyces genomic mini-library is an important strategy to obtain Streptomyces genes,normally used vectors suffer the disadvantage of some false positive percentage,so a high efficient cloning vector would be of practical importance.Methods Through overlap extension PCR,a 96 bp multiple cloning site region was inserted between the promoter and open reading frame of rpsL gene,the DNA fragment was cloned into pBluescript KS（-）,obtaining a new positive selection vector pLS975.Results pLS975 has eighteen cloning sites,recombinant DNA is selected by both ampicillin and streptomycin selection in a streptomycin resistant host strain such as Escherichia coli DH10B.pLS975 showed high cloning efficiencies（96%~100%） in the preparation of two genomic mini-libraries.Conclusion pLS975 can be a good cloning vector for the preparation of Streptomyces genomic mini-library.