副溶血弧菌基因回补实验方法的建立与应用  

Establishment of a method for gene complementation in Vibrio parahaemolyticus

作　　者：陈振鸿[1] 王丽[2,3] 张义全[3] 冯娇[3] 杨瑞馥[3] 常德[1] 安莉[1] 刘长庭[1] 周冬生[3] 

机构地区：[1]解放军总医院南楼呼吸科,北京100853 [2]重庆医科大学检验医学院,重庆400016 [3]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

出　　处：《南方医科大学学报》2014年第1期70-74,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(31170127;81200059);国家重点基础研究发展计划973项目(2014CB744400)~~

摘　　要：目的建立以pBAD33质粒为载体的副溶血弧菌(Vibrio parahaemolyticus,VP)基因回补实验平台。方法 PCR扩增aphA和opaR的整个ORF区序列,并将其直接克隆入pBAD33质粒中,构建重组质粒。分别将重组质粒转入到ΔopaR和ΔaphA中(分别代表opaR和aphA的突变株),以构建出相应的回补株C-ΔaphA和C-ΔopaR。分别在aphA和opaR基因内设计特异性引物,采用RT-PCR方法,验证在相应的回补突变株中aphA和opaR是否转录。利用引物延伸实验研究野生株(WT)、ΔopaR、ΔaphA、C-ΔaphA和C-ΔopaR中mfpA(aphA负调控,opaR正调控其表达)的相对RNA水平。结果 aphA和opaR在相应的回补株中发生转录;且mRNA水平与野生株一致。结论成功建立了以pBAD33质粒为载体的VP基因回补方法,并得到应用。Objective To establish a method for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33. Methods The entire coding region of opaR or aphA was amplified by PCR and cloned into pBAD33. The recombinant plasmid was transformed into AopaR and AaphA （the opaR or aphA null mutant strain, respectively） separately to construct the complemented mutant strain C-△aphA and C-△opaR, respectively. RT-PCR was used to verify the transcription of apaR and aphA in the corresponding complemented mutant strains. Primer extension experiments were performed to determine the relative mRNA levels of mfpA （a gene previously characterized to be negatively regulated by AphA and positively by OpaR） in the wild-type strain, △opaR, AaphA, C-△aphA, and C-△opaR. Results opaR and aphA were transcribed in the corresponding complemented mutant strains, and their mRNA levels were comparable to those detected in the wild-type strains. Conclusion A method has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.

关 键 词：副溶血弧菌 基因回补 pBAD33 aphA opaR 

分 类 号：Q78[生物学—分子生物学]