Expression Of Equine Macrophage CD63 And Preparation Of Monoclonal Antibody

Macrophages are an important part of the body's immune system and one of the target cells of viral infection.The surface molecules are diverse.Studying the interaction of surface molecules is of great significance for the study of viruses.It is particularly important to prepare monoclonal antibodies with specific recognition proteins by immunological interaction with macrophages.CD63 is a surface protein of macrophages and a member of the four-transmembrane protein family.It has four domains,717 bases in total,encoding 239 amino acids,and its theoretical molecular weight is 25.6 kDa.According to the website analysis,there is no signal peptide in this area and it has four transmembrane structures.This protein has been shown to play an important role in the replication of HIV-1 in human macrophages,and CD63 plays a tumor suppressing role in human melanoma.In order to obtain a specific equine CD63 monoclonal antibody,the CD63 protein was purified by prokaryotic and eukaryotic expression systems,the animal is immunized by DNA immunization to stimulate the immune response,and finally a CD63-specific monoclonal antibody is prepared.During the study,it was found that the full length of CD63 could not be expressed in vitro.After transmembrane region software analysis,the sequence of the transmembrane region was removed,and the extracellular region of 297 bases of CD63 sequence(truncated CD63)was selected for prokaryotic expression.The soluble protein was successfully obtained after optimization of the conditions,but the purification was difficult due to the small amount of protein expression.Further adopting the method of eukaryotic expression,the strategy of constructing the recombinant plasmid is to insert the signal peptide with the induced protein secretion and the His tag sequence into the front and the back of the target gene,respectively,and connect to the VR1012 vector,and transfer the obtained positive recombinant plasmid.293 T cells were stained.After 48 hours,the cells and supernatant were collected.By immunoblotting(Western blotting)analysis,soluble expression of CD63 protein was successfully obtained.In order to improve the specificity of protein purification,serum-free medium was selected to be replaced 6 hours after transfection,purified by His tag,and a relatively pure 0CD63 protein was obtained.When the DNA is immunized to the animal,the recombinant expression vector expressing the CD63 protein is injected into the mouse by intramuscular injection,and the mouse is subjected to an immune response reaction,and the animal with high titer is used for the boost test,and the eyeball is used for three days after the boosting.The mice were sacrificed by blood sampling,and the spleens of the mice were fused with myeloma cells to screen for monoclonal antibodies specifically recognizing horse CD63.The specific monoclonal antibody plays an important role in the analysis of intermolecular interactions.The preparation of horse CD63 monoclonal antibody lays a foundation for studying the mechanism of immunoprotection of horse CD63 molecule,and also studies the interaction between horse CD63 and virus.The molecular mechanism provides an effective tool.