〔Abstract〕 Objective To prepare and identify CS-PLGA microspheres carrying bilayer antigen protein.Methods Porous microspheres were prepared by emulsion polymerization combined with solvent evaporation.The antigen was loaded in a water bath at 4 ℃,and the antigen was promoted to enter the microspheres by physical diffusion mediated by antigen concentration gradient and combined with the outer surface of the microspheres by electrostatic action, forming an inner and outer double-layer antigen carrier.Then, according to the glass transition temperature of PLGA material, the pores on the surface of porous microspheres were promoted to heal at 48 ℃ to form a closed microsphere preparation, and then the obtained microsphere preparation was suspended with chitosan solution for cationic modification to reverse the negative potential on the surface of microspheres. In this study,the morphology,particle size distribution and potential changes of microspheres were detected by scanning electron microscope and dynamic light scattering particle size analyzer. In addition,sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE) was used to identify whether the antigen was loaded into the microsphere preparation. The fluorescent labeled BSA and fluorescent labeled PLGA materials were used to observe the distribution of the antigen after loading by laser confocal microscope. The encapsulation efficiency and drug loading rate of the microsphere preparation were detected by BCA method. Results The results of scanning electron microscope and optical microscope showed that the porous microspheres had good pore formation,the particle size was( 73. 94 ± 0. 81) nm,and the Polydispersity index was 0. 038 ± 0. 004. Zeta potential changed from negative to positive,which indicated that chitosan had been successfully coated on the surface of microspheres. SDS-PAGE,laser confocal microscope and other detection methods confirmed that BSA had been successfully carried. The encapsulation rate of porous microspheres was( 3. 01 ± 0. 04) % and the drug loading rate was( 1. 50 ± 0. 02) % after detection by micro BCA kit. Conclusion CS-PLGA preparation carrying bilayer antigen was successfully prepared,which provided a new idea for the subsequent study of sustained and controlled release preparations.