鼠疫耶尔森菌基因组无痕修饰方法的建立  

Development of a high-efficient scarless genetic modification method for Yersinia pestis

作　　者：肖丽生 祁芝珍[3] 吕瑞辰[2] 宋凯[2] 陈荣[2,4] 王敏[2] 吴海莲[3] 赵海红[3] 宋亚军[1,2] 

机构地区：[1]安徽医科大学,合肥230032 [2]军事医学科学院微生物流行病研究所、病原微生物生物安全国家重点实验室,北京100071 [3]青海省地方病预防控制所,西宁811602 [4]解放军总医院微生物科,北京100853

出　　处：《军事医学》2017年第3期209-212,221,共5页Military Medical Sciences

基　　金：国家自然科学基金面上资助项目(31360034,31470138)

摘　　要：目的构建鼠疫耶尔森菌(鼠疫菌)中基因组无痕修饰的技术平台,深入研究鼠疫菌相关基因的功能及作用机制。方法通过不对称PCR扩增抗性盒片段(含修饰靶标区域上下游同源臂),将其导入含pKD46质粒的鼠疫菌中。在阿拉伯糖的诱导下,pKD46质粒可表达与同源重组相关的3个酶(Exo,Beta和Gam蛋白),诱导重组后再导入p KSI-1质粒与目的基因的重组载体,与抗性盒进行第二次同源重组。通过加入阿拉伯糖和IPTG诱导pREDTKI表达重组酶和I-SceⅠ酶,可对未发生重组的抗性盒片段和p KSI-1质粒上的I-SceⅠ位点酶切,从而起到消除抗性盒与质粒作用,达到无痕修饰的目的。结果与结论成功构建了鼠疫菌的Δwaa A和waa A(△9nt)两个突变株。根据不同的实验目的选择相应的基因敲除方法,得到的无痕修饰菌株为鼠疫菌相关基因的功能研究奠定了基础。Objective To construct a technical platform for scarless gene modification of Yersinia pestis and to study the functions of its specific genes. Methods The resistance fragment,including upstream and downstream homologous arms of targeted regions,was reamplified by asymmetric PCR. The amplicons were introduced into Y. pestis harboring plasmid p KD46. With the induction of L-arabinose,the recombinant related enzymes： Exo,Beta and Gam,were expressed to guide the homologous recombination. A donor plasmid,p KSI-1,which carried the desired modification fragment flanking by I-SceⅠrecognition sites,was introduced into Y. pestis as the second step of λ-Red recombination with the help of pREDTKI.Results and Conclusion Two mutant strains： △waa A and waa A（ △9nt）,were successfully constructed for Y. pestis strain201. Scarless modification introduces no extra modification to the genome,and it is ideal for comprehensive functional genomic studies.

关 键 词：鼠疫菌 同源重组 基因编辑 

分 类 号：R378[医药卫生—病原生物学]