细菌CRISPR/Cas免疫系统的构建及功能鉴定  

Recombinant plasmid construction and functional identification of Bacterial CRISPR/Cas9 system

作　　者：刘五高[1] 丁友法[1] 刘爱霞[1] 黄黎丽[1] 金晶[1] 李金美[1] 王伟[1] 

机构地区：[1]丽水市人民医院检验科,浙江丽水323000

出　　处：《中国微生态学杂志》2015年第9期1031-1033,共3页Chinese Journal of Microecology

摘　　要：目的构建细菌CRISPR/Cas免疫系统表达质粒,用于细菌耐药基因水平转移的研究。方法 PCR技术分三段扩增嗜热链球菌LMD-9的CRISPR/Cas基因序列,并依次接入PACYCDuet-1质粒。以体外酶切效率评价其活性。结果经PCR扩增、测序鉴定,成功扩增了完整的CRISPR/Cas系统;在功能活性鉴定中,gRNA剪切靶点DNA酶切条带的灰度值分别为10和3,表明构建的CRISPR/Cas系统质粒具有活性。结论成功构建了细菌CRISPR/Cas免疫系统质粒,为今后用于细菌耐药基因水平转移打下了基础。Objective To construct the expression plasmid of bacterial CRISPR/Cas immune system for use in studies on horizontal transfer of resistant gene. Methods The full-length CRISPR/Cas system from Streptococcus thermophilus LMD-9 was amplified by PCR and cloned into PACYCDuet-1 vector followed by identification with sequencing. The activity was identified by cutting efficiency of Cas/gRNA to PMG252 plasmid in vitro. Results The amplification of CRISPR/Cas system was verified by PCR and DNA sequencing identification. The gray value of gRNA shear digested DNA bands were 10 and 3, respectively, indicating that the constructed CRISPR/Cas system has activity. Conclusion The bacterial CRISPR/Cas immune system plasmids were successfully constructed, which provides the basis for future horizontal transfer of bacterial resistant gene.

关 键 词：CRISPR/Cas9系统 质粒 细菌 

分 类 号：S852.4[农业科学—基础兽医学]