南方医科大学学报
南方醫科大學學報
남방의과대학학보
JOURNAL OF SOUTHERN MEDICAL UNIVERSITY
2014年
1期
70-74
,共5页
陈振鸿%王丽%张义全%冯娇%杨瑞馥%常德%安莉%刘长庭%周冬生
陳振鴻%王麗%張義全%馮嬌%楊瑞馥%常德%安莉%劉長庭%週鼕生
진진홍%왕려%장의전%풍교%양서복%상덕%안리%류장정%주동생
副溶血弧菌%基因回补%pBAD33%aphA%opaR
副溶血弧菌%基因迴補%pBAD33%aphA%opaR
부용혈호균%기인회보%pBAD33%aphA%opaR
Vibrio parahaemolyticus%gene complementation%pBAD33%aphA%opaR
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。
目的:建立以pBAD33質粒為載體的副溶血弧菌(Vibrio parahaemolyticus, VP)基因迴補實驗平檯。方法PCR擴增aphA和opaR的整箇ORF區序列,併將其直接剋隆入pBAD33質粒中,構建重組質粒。分彆將重組質粒轉入到ΔopaR和ΔaphA中(分彆代錶opaR和aphA的突變株),以構建齣相應的迴補株C-ΔaphA和C-ΔopaR。分彆在aphA和opaR基因內設計特異性引物,採用RT-PCR方法,驗證在相應的迴補突變株中aphA和opaR是否轉錄。利用引物延伸實驗研究野生株(WT)、ΔopaR、ΔaphA、C-ΔaphA和C-ΔopaR中mfpA(aphA負調控,opaR正調控其錶達)的相對RNA水平。結果 aphA和opaR在相應的迴補株中髮生轉錄;且mRNA水平與野生株一緻。結論成功建立瞭以pBAD33質粒為載體的VP基因迴補方法,併得到應用。
목적:건립이pBAD33질립위재체적부용혈호균(Vibrio parahaemolyticus, VP)기인회보실험평태。방법PCR확증aphA화opaR적정개ORF구서렬,병장기직접극륭입pBAD33질립중,구건중조질립。분별장중조질립전입도ΔopaR화ΔaphA중(분별대표opaR화aphA적돌변주),이구건출상응적회보주C-ΔaphA화C-ΔopaR。분별재aphA화opaR기인내설계특이성인물,채용RT-PCR방법,험증재상응적회보돌변주중aphA화opaR시부전록。이용인물연신실험연구야생주(WT)、ΔopaR、ΔaphA、C-ΔaphA화C-ΔopaR중mfpA(aphA부조공,opaR정조공기표체)적상대RNA수평。결과 aphA화opaR재상응적회보주중발생전록;차mRNA수평여야생주일치。결론성공건립료이pBAD33질립위재체적VP기인회보방법,병득도응용。
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 ΔopaR and ΔaphA (the opaR or aphA null mutant strain, respectively) separately to construct the complemented mutant strain C-ΔaphA and C-ΔopaR, respectively. RT-PCR was used to verify the transcription of opaR 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, ΔopaR, ΔaphA, C-ΔaphA, and C-ΔopaR. 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.