Construction Of Fc Gamma RⅡB Lentivirus Vector And Preliminary Investigation Of Its Regulation To The Antigen Presentation Of ImDC Cells In Rats

Objective: To construct the expression vector for lentivirus-induced FcγRⅡB in rats and to test whether the expression vector of FcγRⅡB lentivirus can be stably and controllably expressed on the surface of HT-1080 cells.After the immature dendritic cells were transfected,the effect on antigen presenting function was studied.Methods: FcγRIIB gene fragment was reverse transcribed using Lewis rat m RNA as a template.The FcγIIB gene fragment was cloned and inserted into lentiviral plasmid TRE to construct the recombinant plasmid expressing FcγRIIB lentivirus.The expressed virus was harvested by transfecting HEK293 T cells with the FcγRⅡB lentiviral recombinant plasmid and the Lenti-X HTX lentiviral packaging system.Lentivirus regulatory plasmid Tet-on3 G and Lenti-X HTX packaging system was transfected into HEK293 T cells to harvest the regulatory virus.HT-1080 cells were co-infected with the expression and control viruses.The immunofluorescence staining was used to observe the expression of Fcy RIIB in HT-1080 cells induced by the doxorubicin(Dox)gradient.The immature dendritic cells(im DC)were cultured in vitro.Flow cytometry was used to identify the cell phenotype and cell purity quotient.The expression of virus and the regulatory virus co-infected immature dendritic cells.Dox was added to im DC Immunofluorescence staining was used to observe the expression of FcγRⅡB on the surface of im DC cells.Western blot was used to detect the expression of FcγRⅡB at the post-transcriptional level.Flow cytometry was used to detect the expression of CD80,CD86,CD40 and MHC-II on the cell surface Expression on the surface of im DC cells after transfection.Results: The results of immunofluorescence staining showed that FcγRIIB could be controllably expressed in HT-1080 cells.Before transfection Flow cytometry results showed that the positive rate of OX62 was 93% on im DC.Flow cytometry showed that the im DC which was transfected positive rates of CD80,CD86,CD40 and MHC-II were 11.6% and 11.48%,9.25% and 12.35% respectively.Immunofluorescence results showed that im DC cells could up-regulate the expression of FcγRⅡB on the surface of the cells after transfection.The expression of FcγRⅡB which was expressed on the tranfected im DC by flow cytometry was positively correlated with the dosage of inducer.After im DC was transfected,the Western blot results also showed the expression of proten FcγRⅡB was positively correlated with the dosage of inducer.The positive expression rates of CD80,CD86,CD40,and MHC class I,I were 6.44%,8.25%,5.38% and 11.62% respectively by flow cytometry after transfection on im DC.Conclusion: The FcγRIIB lentivirus-induced expression vector was successfully constructed.The lentivirus infects rat bone marrow by way of derived immature dendritic cells,and the overexpressed FcγRIIB gene,which can deregulate the antigen presentation of immature dendritic cells.