中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2014年
2期
149-153
,共5页
叶美萍%黄龙丽%庄振超%楼永良
葉美萍%黃龍麗%莊振超%樓永良
협미평%황룡려%장진초%루영량
伯氏疏螺旋体%穿梭载体%诱导表达%工具质粒
伯氏疏螺鏇體%穿梭載體%誘導錶達%工具質粒
백씨소라선체%천사재체%유도표체%공구질립
Borrelia burgdorferi%Shuttle vector%Induced expression%Tool plasmid
目的 构建一个具有较强遗传可操作性的大肠杆菌-伯氏疏螺旋体表达穿梭质粒,以期作为工具质粒在疏螺旋体中实现外源基因的可诱导表达.方法 利用源自大肠杆菌的lac表达/诱导系统,在已有的穿梭质粒的基础上,通过分子生物学技术加入多克隆位点和HA、FLAG蛋白表达标签,增加其遗传可操作性.结果 成功构建工具质粒pJ J275,可以用于伯氏疏螺旋体的基因可控制表达.以rpoS基因为例,将其克隆至pJJ275并转入疏螺旋体中,获得的菌株在IPTG存在的条件下可诱导表达目的蛋白RpoS及其控制下的OspC.结论 构建的工具质粒易于分子生物学操作,可以实现疏螺旋体中外源基因的可控制表达.
目的 構建一箇具有較彊遺傳可操作性的大腸桿菌-伯氏疏螺鏇體錶達穿梭質粒,以期作為工具質粒在疏螺鏇體中實現外源基因的可誘導錶達.方法 利用源自大腸桿菌的lac錶達/誘導繫統,在已有的穿梭質粒的基礎上,通過分子生物學技術加入多剋隆位點和HA、FLAG蛋白錶達標籤,增加其遺傳可操作性.結果 成功構建工具質粒pJ J275,可以用于伯氏疏螺鏇體的基因可控製錶達.以rpoS基因為例,將其剋隆至pJJ275併轉入疏螺鏇體中,穫得的菌株在IPTG存在的條件下可誘導錶達目的蛋白RpoS及其控製下的OspC.結論 構建的工具質粒易于分子生物學操作,可以實現疏螺鏇體中外源基因的可控製錶達.
목적 구건일개구유교강유전가조작성적대장간균-백씨소라선체표체천사질립,이기작위공구질립재소라선체중실현외원기인적가유도표체.방법 이용원자대장간균적lac표체/유도계통,재이유적천사질립적기출상,통과분자생물학기술가입다극륭위점화HA、FLAG단백표체표첨,증가기유전가조작성.결과 성공구건공구질립pJ J275,가이용우백씨소라선체적기인가공제표체.이rpoS기인위례,장기극륭지pJJ275병전입소라선체중,획득적균주재IPTG존재적조건하가유도표체목적단백RpoS급기공제하적OspC.결론 구건적공구질립역우분자생물학조작,가이실현소라선체중외원기인적가공제표체.
Objective To construct a shuttle plasmid for inducible gene expression in Borrelia burgdorferi (B.burgdorferi) with an advantage of flexible genetic manipulation.Methods The IPTG-inducible lac repressor/operator system from Escherichia coli (E.coli) was adopted and modified in the current study.The plasmid shuttle vector was developed by inserting multiple cloning sites,FLAG and HA tags into the shuttle vector by molecular cloning approaches.The target gene was inserted at the site under the control of the promoter (Tn5 derivate) in plasmid pQE30.This promoter contained two lac operators and a codonoptimized lacI gene driven by flaB promoter.Results A plasmid shuttle vector,pJJ275,was successfully constructed with the ability to express target genes in B.burgdorferi in the presence of IPTG.By using this system,a HA-tagged rpoS gene was introduced into the typical infectious strain B.burgdorferi B31.The target gene expression induced by IPTG was confirmed at transcriptional and translational levels.The RpoS dependent virulence factor of Borrelia,OspC,was also detected,indicating that the expressed protein was functional.Conclusion The constructed plasmid shuttle vector can express exogenous genes in B.burgdorferi with an inducible feature and an advantage of flexible genetic manipulation.It can be applied for genetic manipulation of B.burgdorferi involved in gene regulation and complementation.
