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AIM:Bacillary dysentery caused by Shigella flexneri is stilla threat to human health.Of four invasion plasmid antigenproteins (IpaA,B,C and D),IpaC plays an important role inthe pathogenicity of this pathogen.The purpose of this studywas to investigate the proteins interacting with IpaC in thehost cell during the pathogenic process of this disease.METHODS:By applying two-hybrid system,the baitplasmid containing ipaC gene was constructed anddesignated pGBKT-ipaC.The bait plasmid was transformedAH109,and proved to express IpaC and then HeLa cDNAlibrary plasmids were introduced into the above transformedAH109.The transformation mixture was plated on mediumlacking Trp,Leu,and His in the initial screen,then restreakedon medium lacking Trp,Leu,His and Ade.Colonies growingon the selection medium were further assayed for β-galactosidase activity.BLAST was carried out in thedatabase after sequencing the inserted cDNA of the positivelibrary plasmid.RESULTS:Among the 2×10~6 transformants,64 positiveclones were obtained as determined by activation of His,Ade and LacZ reporter genes.Sequence analysis revealedthat cDNA inserts of two colonies were highly homologousto a known human protein,RanBPM.CONCLUSION:These results provide evidence that IpaCmay be involved in the invasion process of S.flexneri byinteracting with RanBPM,and RanBPM is most likely to bethe downstream target of IpaC in the cascade events of S.flexneri infection.
AIM: Bacillary dysentery caused by Shigella flexneri is stilla threat to human health. Of four invasion plasmid antigen proteins (IpaA, B, C and D), IpaC plays an important role in pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease. METHODS: By applying two-hybrid system, the baitplasmid containing ipaC gene was constructed anddesignated pGBKT-ipaC.The bait plasmid was transformedAH109, and proved to express IpaC and then HeLa cDNAlibrary plasmids were introduced into the above transformedAH109.The transformation mixture was plated on mediumlacking Trp, Leu, and His in the initial screen, then restreakedon medium lacking Trp, Leu, His and Ade .Colonies growingon the selection medium were further assayed forβ-galactosidase activity. BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid .RESULTS: Among the 2 × 10 ~ 6 transformants, 64 pos Itiveclones were obtained as determined by activation of His, Ade and LacZ reporter genes. Sequence analysis revealed that cDNA inserts of two colonies were highly homologousto a known human protein, RanBPM.CONCLUSION: These results provide evidence that IpaCmay be involved in the invasion process of S .flexneri byinteracting with RanBPM, and RanBPM is most likely to be downstream goal of IpaC in the cascade events of S.flexneri infection.