Construction Of Fluorescent Protein Expression Vectors Respectively Driven By Promoters Of F-ATPase And RecA

Objective: F-ATPase(membrane-bound proton-translocating ATPase, F₀F₁ATPase), an important aciduric factor of caries pathogen, is essential prerequisite for cariogenesis. As a major component of acid-adaptive response mechanism of cariogenic bacteria, F-ATPase makes bacteria keep activity even in low pH biofilms environment through maintaining internal pH values by pumping protons out of cells. Meanwhile, with more and more protons excretion and accumulation, the pH value in dental plaque reaches 5.4 or 5.5 and results in dental caries. Therefore, F-ATPase plays a key role in dental caries. It is well known that both phenotype characteristics and gene expression of cariogenic pathogens in biofilms are different from in planktonic status. Also it is undoubted that only in the dental pique biofilms can the pathogens performance their cariogenic function. In order to illustrate the F-ATPase's role of the cariogenic pathogens in dental plaque, we constructed a green fluorescent protein expression vector driven by promoter of F-ATPase operon of Streptococcus mutans(S.mutans), and a red fluorescent protein shuttle vector promoted by RecA operon's promoter as a control.Methods:1. Construction of the green fluorescent protein expression vector driven by F-ATP operon promoter:The promoter sequence of F-ATPase operon amplified from PCR was inserted into a plasmid express vector pEGFPN1 to construct a Prokaryotic expression vector pFgfp. Then the promoter gene of F-ATPase operon and a green fluorescent protein gene were ligated together into a shuttle vector pDL276 named as pLFgfp.2. Construction of the red fluorescent protein expression vector driven by RecA operon promoter: pRred, a recombinant vector containing RecA operon promoter which was separated from T vector, was constructed and transformed into DH5α. After that the red fluorescent protein gene promoted by RecA operon promoter, separated from pRred, was inserted into a shuttle vector pDL276, named as pLRred.3. Transformed into host cell-UA159:These above two shuttle vectors were transformed into S. mutans and assayed with fluorescence microscopy.Results:1. pLFgfp, the recombinant shuttle vector containing a green fluorescent protein promoted by F-ATPase operon promoter, expressed green fluorescent protein in DH5a cells.2. pLRred, containing RecA operon promoter and red fluorescent protein gene, was successfully constructed and -expressed red fluorescent protein in cells.3. Both of the two vectors respectively containing F-ATPase and RecA promoters were separately transformed into the host cells S.mutans and express the fluorescent proteins.Conclusion: The recombinant vectors pLFgfp and pLRred were successfully constructed, which provide an important foundation for further study on relationship between acid-adaptive response of S. mutans F-ATPase and cariogenesis under different pH condition in biofilms.