The Recombinant Adenovirus Of CTLA4Ig And α4β7 Modify Dendritic Cells

PARTⅠTHE CLONE OF INTEGRINα4 GENE AND INTEGRINβ7 GENE OF THE MICEObjective: To clone the integrinα4 gene and integrinβ7 gene from the intestine peyer's patches(PP) of the miceMethods: Total RNAs were isolated from the intestine PP of the mice. The integrinα4 gene and integrinβ7 gene were amplified by RT-PCR using the primers based on the published sequence ofα4,β7. The PCR products were purified by gel extraction and cloned into pMD-18, pMD-19. Analyzed the recombinant plasmids pMD-18-α4,pMD-19-β7 through sequencing and sequence comparison with theα4,β7 genes in GenBank, repaired the basic groups which developed site-specific mutagenesis.Results: Theα4,β7 genes were cloned. 6 basic groups inα4 gene order and 5 basic groups inβ7 gene order by the sequence comparison were revealed, which developed site-specific mutagenesis and resulted in encoding alteration. The mutational basic groups resulting in encoding alteration with site-specific mutagenesis technology were repaired. Conclusion: The integrinα4,β7 genes were cloned in mice and the basic groups which developed site-specific mutagenesis were repaired.PARTⅡCONSTRUCTION OF THE RECOMBINANT ADENOVIRUS : ADCTLA4IG,ADα4β7Objective: To construct the recombinant adenovirus: AdCTLA4Ig,Adα4β7, including CTLA4Ig,α4,β7 genes and controls AdGFP with the newest AdEasy System, to clone IRES series from pIRES2-EGFP , link and insertα4,β7 genes to the same gene expressing frame.Methods: 1.The CTLA4Ig genes were coloned to the shuttle plasmid pAdTrack-CMV. The linearized shuttle plasmids were co-transformed with adenoviral backbone vector to E.coli BJ5183 cells. Before transfection the recombinant adenoviral plasmids were digested with PacI. Then the digested recombinant adenoviral plasmids were transfected to 293 cells with Lipofectamine 2000. The generations of the recombinant adenoviruses were monitored by GFP expression. The presences of recombinant adenoviruses were further confirmed by PCR. 2. Theα4,β7 genes,IRES series were coloned to the shuttle plasmid pAdshuttle-CMV. The linearized shuttle plasmids were co-transformed with adenoviral backbone vector to E.coli BJ5183 cells. Before transfection the recombinant adenoviral plasmids were digested with PacI. Then the digested recombinant adenoviral plasmids were transfected to 293 cells with Lipofectamine 2000. The constructions were confirmed with PCR and gene expressions were defined with Western Blot.Results: 1. The straps of 4.5kb and near 30kb appeared after digested with Pac I. The results showed that the fusion genes encoding CTLA4Ig to the shuttle plasmid pAdTrack-CMV were cloned. The correct recombinant adenoviral plasmids constructed were conformed by the restriction analysis and the PCR technique. And the fluorescence was observed in 293 cells. 2. The straps of 4.5kb and near 30kb appeared after digested with PacI .The results showed that integrinα4 gene and integrinβ7 gene were cloned to the shuttle plasmid pAdshuttle-CMV. The restriction analysis and the PCR conformed that correct recombinant adenoviral plasmid was constructed. Western Blot indicated expression of objective genes.Conclusion: The recombinant adenovirus of AdCTLA4Ig,Adα4β7 and controls AdGFP were constructed. PARTⅢTRANSDUCT ADCTLA4IG AND ADα4β7 INTO DENDRITIC CELLSObjective: The dendritic cells were isolated from the bone marrow of the mice to set up the primary culture method in vitro. To identify the factors that influenced the mature of the dendritic cells and the expression efficiency of CTLA4Ig andα4β7. To construct the dendritic cell vaccines transducted by AdCTLA4Ig and Adα4β7.Methods: The bone marrow cells were collected from the tibial and femurs of the mice, and cultured with GM-CSF and IL-4 in vitro for 7 days. The growth and morphology of the cells were observed by light microscope. The dendritic cells were identified by FACS and transfected with the recombinant adenovirus, and then cocultured with OVA. The maturation of the dendritic cells was identified by FACS. The expression of intracellular report gene and the growth of the dendritic cells were detected by fluorescence microscope. The expressions of purpose genes were detected by Western blotting and the expressions of CTLA4Ig andα4β7 proteins in the same cells were detected by laser scanning confocal microscope. The dendritic cells and T lymphocytes were co-cultured, then the impact on the proliferation and the apoptosis of T lymphocytes from the dendritic cells were detected by MTT and TUNEL, respectively. Results: 50% of the dendritic cells were showed the phenotype of CD11c and were definited as the dendritic cells. The proteins in the infected cells were extracted and detected. The positive protein bands of CTLA4Ig andα4β7 were observed on the location of 53kDa and 198kDa, respectively. About 50% of the cells coexpressed CTLA4Ig andα4β7 proteins. The dendritic cells that coexpressed CTLA4Ig andα4β7 proteins suppressed the proliferation of the T lymphocytes and promoted the apoptosis of T lymphocytes.Conclusion: The transduction of AdCTLA4Ig and Adα4β7 into dendritic cells could make the dendritic cells coexpress CTLA4Ig andα4β7 proteins. The expressions of costimulatory molecules CD86 and CD80 on the dendritic cells were decreased. The dendritic cells could suppress the proliferation and promoted the apoptosis of T lymphocytes.