Research On The Role Of Rab5 And Rab7 In Cross Presentation

Cross-presentation is the process by which extracellular antigens were presented by MHC classâ… molecules.This differs from the classical MHC classâ… processing pathway. Classical MHC classâ… antigen presentation begins with the degradation of intracellularly synthesized proteins by the proteasome.A fraction of the peptide fragments that result from this degradation is transported into the ER by the peptide transporter TAP(transporter associated with antigen presentation).In the ER,the peptides are loaded onto newly synthesized MHC classâ… molecules,and these complexes are then transported to the cell surface.In contrast,the MHC classâ…¡processing pathway is dedicated to the presentation of exogenous antigens that are degraded in the endocytic pathway.MHC classâ… and MHC classâ…¡molecules thus sample antigenic information from different sources,intracellular and extracellular antigens,respectively.In vivo,DCs,the major cell type responsible for cross-priming-acquire endogenous antigens from infected cells in the periphery,and then migrate to the lymph nodes where they display antigenic peptides in association with MHC classâ… molecules.MHC classâ… -peptide complexes are recognized by antigen-specific CTLs,which become activated and expand in response to antigen recognition.In this scenario,the source of antigens (intracellular,but from a different cell,or extracellular as in vaccination settings) is distinct from that usually sampled by the classical MHC classâ… antigen presentation pathway (intracellular antigens within the antigen presenting cell).Hence the mechanism of antigen degradation and delivery of the peptide to MHC classâ… molecules is also likely to be different.The mechanism of cross-presentation has arose much interest in recent years,in part because cross-presentation is likely to be important in activating CTLs in response to vaccine antigens.Intracelluar trafficking of membrane proteins requires a complex of proetains that include members of the Rab family.Rab GTPases are key to membrane-trafficking events in eukaryotic cells.The human genome encodes almost 70 Rabs and Rab-like proteins,and members of this large family of Raslike GTPases are localized to distinct membrane-bound compartments.Rabs use the guanine nucleotide-dependent switch mechanism common to the superfamily to regulate each of the four major steps in membrane traffic:vesicle budding,vesicle delivery,vesicle tethering,and fusion of the vesicle membrane with that of the target compartment.Accumulated evidence shown that several rab proteins have been localized to early sorting and recycling endosomal compartments(Rab4,Rab5,Rab11, Rab18,Rab22 and Rab25) and Rab7 and Rab9 have been localized to late endosomes. However,to date little has been done to examine the function of Rab proteins in the antigen cross presentation of dendritic cell.The primary goal of this study is to determine the role of Rab5 and Rab7 antigen cross presenentation by transiently over expressing several mutants in dendritic cell line DC2.4. Our results indicate that over expression the dominnat-negative Rab5 mutatn(Rab5S34N) inhibit the cross presentation of exogenous antigen E Coli in dendritic cell,whereas expression of the corresponding Rab7 mutant(Rab7T22N) and constitutively active Rab5 mutant(Rab5Q79L) does not.The data propose the early endosome is the major compartment in which exogenous antigen degradation and cross presentation.Objection:Try to research on the role of Rab5 and Rab7 in coross presentation,we made and express the fusion protein dsRed tagged OVA antigen via construction an expression vector pET16bOR and pET16bdsRedOVA.They can be used for further functional analysis for antigen presentation.We also made Rab5 and Rab7 mutations,it provides us a chance to studay the function of Rab5 and Rab7 in cross-presntation.Method:To achieve this goal,we conducted the following works:1.The construction and express of the vector pET16bOR and pET16bdsRedOVA.(1).Overlapping oligonucleotides that contain an Ageâ… site and OVA_257-264 encoding sequence were hybridized to a double strand adaptor.The adaptor and dsRed fragment from plasmid pdsRed-C1 were cloned into pET-16b,and then E.coli BL21 cells were transformed with the correct insertion(pET-16bOR) and induced with isopropyl beta-D-thiogalactoside.The dsRed tagged OVA epitope fusion protein is obtained by genetic engineering methods.It keeps the fluorescent characters of dsRed and can be used for further functional analysis for antigen presentation. (2).Overlapping oligonucleotides that contain Notâ… -BamHâ… sites were hybridized to pET-16bOR.The recombinant vector was identified and then named pET16myc.Then the dsRed fragment was cut from plasmid pdsRed-C1 via Ageâ… -Kpnâ… sites was cloned into pET16myc.Then the recombinant vector was named pET16myc.Lastly,the OVA fragment was cut from plasmid pCMV-cyt OVA via Sacâ… and Salâ… sites was cloned into pET16Rmyc, the recombinant vector was identified and named pET16dsRedOVA.2.Cross-presentation of Escherichia coli that express the recombinant protein in DC cells.A plasmid that contains the expression element of fusion protein dsRed tagged OVA was constructed and expressed in E coli.The cross presentation and degradation of the E. Coli.in DC cells were dynamically assessed with T cell hybridoma.3.The construction of Rab5 and Rab7 mutations lentiviral vectors.We designed and constructed the Rab5 and Rab7 mutations lentiviral vectors. (pSIF1-copGFPRab5wt,pSIF1-copGFPRab5Q79L,pSIF1-copGFPRab5S34N pSIF1-copGFPRab7wt and pSIF1-copGFPRab7T22N)4.To observe effect of tha Rab5 and Rab7 mutations vector in cross presentation.To determine the role of Rab5 and Rab7 antigen cross presenentation by transiently over expressing several mutants in dendritic cell line DC2.4.Results&Conclusions:we constructed the vector of dsRed tagged OVA successfully. The dsRed tagged OVA fusion vector was confirmed by restricted enzyme degested and the characteristics of recombinant protein was identified as the same as the fluorescent characters of dsRed.It can be used for further functional analysis the antigen presentation. Furthermore,we made successfully Rab5 and Rab7 mutations lentiviral vectors.At last Our results indicate tha over expression the dominnat-negative Rab5 mutatn(Rab5S34N) inhibit the cross presentation of exogenous antigen E Coli in dendritic cell.