Construction And Evaluation Of The Suicide Plasmid Containing GFP Reporter In Streptococcus Pneumoniae

Streptococcus pneumoniae is the opportunity pathogen which processes transformation more efficiently than other microbes. Therefore, environmental factors are relative to the evolution,variation and pathogenicity of S.pn and we should connect them with the pathogenesis of S.pn. It is important to study S.pn in vivo environment. Priorly, we must find a probe suitable for investigation in vivo. GFP is an ideal probe applied to IVET(in vivo expression technology) because it can be directly detected in vivo. Objective: To use green fluorescent protein to observe pneumococcal adhesion and invasion in vivo and analyze pneumococcal virulence factors. Methods: Gene cloning was used to construct suicide plasmids containing the gfp reporter by which homologous recombination was completed between S.pn DNA and fusion geneply-gfp. With the technology of expression in vivo we compared the expression of GFP in vivo with the result of S.pn'culturing in media and analyzed pneumococcal virulence factors. Results: Restriction maps show that the structures of pEVP3-gfp and pEVP3-ply-gfp are exactly the same as anticipated. Further results indicate that the recombinant S.pn is not different from the un- recombinant S.pn and that both GFP and PLY were simultaneously expressed in S.pn. Conclusion: A new plasmid was established as the vector for studying the expression of pneumococcal virulence factors in vivo. It will facilitate analyzing and screening the candidate virulence factors.