Construction,Expression Of CM614-629/I-A~d/IgG2b Fc Dimer Fusion Protein And Its Identification

Objective: Constructed CM614-629/I-A^d/IgG2b Fc baculovirus expression vector to express CM614-629/I-A^d / IgG2b Fc dimer fusion protein in sf9 insect cells.Methods: I-A^d?,I-A^d? and IgG2b Fc gene sequences were amplified from Bal b/c mouse lymphocytes by RT-PCR.Synthesize the gene sequence of Fos,Jun and the CM614-629 antigen peptide.I-A^d?-Fos recombination sequence was constructed by connecting the leucine zipper sequence of Fos with I-A^d? by using overlapping PCR.CM614-629/I-A^d?-Jun were constructed by connecting CM614-629,I-A^d? and the leucine zipper sequence of Jun via overlapping PCR;The recombinant vector pFastBac^TMDual+[CM614-629/I-A^d/IgG2b Fc] was constructed by inserting I-A^d ?-Fos,CM614-629/I-A^d?-Jun and IgG2b Fc to the Shuttle plasmid,pFastBac^TMDual.We use PCR,restriction endonuclease and Sequencing to identify the recombinant vector.The recombinant plasmid PFastBac^TMDual+[CM614-629/I-A^d/IgG2b Fc]was transferred into the DH10 Bac competent state to form recombinant baculovirus Bacmid+[CM614-629/I-A^d/IgG2b Fc]under the action of the transposon.we transfected the recombinant baculovirus into sf9 insect cells by liposome transfection reagent.After infected with sf9 insect cells,the supernatant was collected by PEG20000 to obtain I-A^d/IgG2b Fc dimer fusion protein.The fusion protein was detected by double-antibody sandwich ELISA and Western-blot.Results: PCR,restriction enzyme digestion and sequencing confirmed that the recombinant vector pFastBac^TMDual+[CM614-629/I-A^d/IgG2b Fc]had the correct sequence.The double antibody sandwich ELISA and Western-blot showed that recombinant bacmid could successfully infect sf9 insect cells,and the expressed fusion protein had the correct conformation.Conclusion: The CM614-629/I-A^d/IgG2b Fc baculovirus expression vector was successfully constructed and expressed in sf9 insect cells,laying a foundation for the study of I-A^d-restricted T cells...