Estabolishment Of HBsAg Target Chimeric Antigen Receptor NK92MI Cells

Objective Prepare hepatitis B surface antigen(HBsAg)targeted chimeric antigen receptor(CAR)NK92MI cells by constructing a lentiviral expression vector of chimeric antigen receptor recognizing HBsAg and express it in NK92 MI cells.Methods 1.Design and synthesis of the CAR: CAR was designed by the sequence of single chain variable fragment(sc Fv)that we previously screened and the structure of CAR described in the literature and synthesized.2.Construction of the recombinant plasmid: Several types of CAR were constructed by molecular cloning and ligated with lentiviral expression vectors to construct recombinant plasmids expressing CAR.3.The packing concentration and titer determination of the virus: The recombinant plasmid and the packaging plasmid were transfected into HEK 293 T cells.Then,the packaged lentivirus particles were concentrated and the virus titer was measured by multiple dilution method.4.Infection of target cells by virus: HEK 293 T cells and NK92 MI cells were infected with a certain amount of concentrated lentivirus.5.Verification of CD3ζ : Verify the expression of CD3ζ in HEK 293 T cells and NK92 MI cells by Western blotting.6.Measurement of infection efficiency: measure the virus infection efficiency in NK92 MI cells by Flow cytometry.Results 1.It showed that CAR was integrated into lentiviral expression vector,and lentiviral expression vector recognizing chimeric antigen receptor of HBsAg was constructed successfully.2.Packing and concentration of lentivirus expression vector were completed,and the titer of lentivirus was measured to be ≥107 transducing units(TU)/ m L.3.The results of Western blotting showed that CD3ζ was successfully expressed in HEK 293 T cells and NK92 MI cells.Conclusion The chimeric antigen receptor NK92MI(CAR-NK92MI)cells recognizing HBsAg were constructed successfully in this research.