A Novel Method For Generation Of Antibody To Protein Based On B Cell Epitope

Antibody is very important for protein research, as it can specifically bind protein, and make researchers identify protein and study the function of protein in vivo and in vitro. With the rapid development of proteomics, a large amount of antibodies are needed for the requirement of high-throughput proteomics research.The whole native protein antigen or synthetic peptide is required for traditional method of antibody generation, but the whole native protein is not always available, and the protein purification is time-consumed and laborious, while the synthesis of peptide through chemical methods is of high cost, and the synthetic peptides should be coupled to protein carrier for animal immunization. So, the acquisition of the antigens is a key rate-limiting step for high throughput generation of antibodies.Antigen epitopes are the basic structural and functional units for specific immune response. So, this paper focuses on the epitopes of antigen, and attempts to develop a new method of generation of antibody to proteins based on the B cell epitopes, and achieves the generation of antibody to proteins without the whole native protein antigen.The strategy of this paper is as followed. Firstly, select the specific B cell epitopes based on the data of protein structure and display the selected epitopes on the T7 phages, then extract the recombinant T7 phages and immunize the animals, and make monoclonal antibody through cell fusion, and finally select the antibody recognizing the source native protein by ELISA or Western blot.Selection the effective B cell epitope of protein antigen is a key to making antibody without whole native protein. If the data of the three dimensional structure of a protein is available, the peptide located in loop or coil region can be selected. If there is no information about three dimensional structure of a protein, six to ten-amino acid peptide in C-terminal of the protein can be selected, and it is better that this C-terminal peptide is specific for the protein. The B cell epitope can also be selected using B cell epitope prediction program, the epotipes predicted by different programs can be selected as candidate epitopes. For proteins with high homology, the protein sequences should be aligned, and the peptides with low homology can be selected. Based on above principles, fourteen epitopes from ten proteins were selected, and twelve of them were testified.The peptide of B cell epitope of protein is small, and is easily degraded when injected in animals. So it should be coupled to protein carrier to increase its immunogenicity. T7Select415-1b vector was used to display the B cell epitope in this paper. This vector can display 415 copies of peptides up to fifty amino acids on the surface of T7 capsid. Twelve epitopes from ten different proteins were displayed on T7 phages using T7Select415-1b vector. The recombinant T7 phages were extracted and used to immunize mice, 6 weeks later, the anti-sera was analyzed by ELISA. The results indicated that all the recombinant phages induced the generation of specific antibody against the respective B cell epitope. Four kinds of anti-sera were analyzed using Western blot, the data showed that the antibodies induced by the 4 epitopes selected from loop regions or C-terminal of the proteins all could recognize its corresponding source native protein. This meant that T7 phage-epitope complex has good immunogenicity, it can be used as good immunogen instead of keyhole limpet hemocyanin or bovine serum albumin coupled peptide antigen.The procedure of mix-immunization using multi-epitopes was tried. Five T7 phage-displayed epitopes from five proteins were mixed and used to immunize rabbit, eight weeks later, the anti-sera was analyzed. The results indicated that the anti-sera could recognize all the five epitopes. But the antibody titers were different. With the increasing of immunization times, the titers of antibody were decreasing gradually. Only the titer of antibody against MAGE A10 epitope was always at a high level, others all decreased dramatically. This meant that the MAGE A10 epitope maybe a dominant epotipe among the five epitopes. In order to obtain the desired anti-sera using mix-immunization strategy, the times of immunization should be controlled, and the opportunity to collect the anti-sera should be grasped. To separate specific antibody against a certain epitope from sera, two epitope-coupled affinity columns were prepared. Antibody mixture was separated by series affinity columns. Two kinds of specific polyclonal antibody were obtained by respective eluting of the two affinity columns. This study laid the foundation in preparation of bulk of mono-specific polyclonal antibody.To further confirm the feasibility of the new antibody preparation method with alternative antigen strategy based on B cell epitope, the specific monoclonal antibodies against three protein antigens were generated. Three B cell epitopes were selected from HPO, Pif1αand Pif1β, and displayed on T7 phages. After immunization with the recombinant T7 phages, the spleen cells were used to fuse with SP2/0 cells, and finally three hybridoma cells, which secreted antibody against the corresponding epitope, were obtained. If the monoclonal antibody against Pif1βepitope could recognize Pif1βprotein was not confirmed because of lacking whole protein of Pif1β. The monoclonal antibodies directed against the epitopes from HPO and Pif1αall could specifically recognized its corresponding source native protein analyzed by Western blot. The monoclonal antibody against the epitope of Pif1αwas further used to identify the subcellular localization of Pif1αusing immuno fluorescence, and the result was consistent with the reported results in literature. These data indicate that the new method for generation of antibody to protein based on B cell epitope is fully feasible.In conclusion, a new method for generation of antibody to protein based on B cell epitope was developed in this paper. This new method can not only carry out the rapid preparation of alternative antigens based on B cell epitope, but also meet the need of antigens in the high-throughput antibody generation in proteomics studies. On the other hand, this method can also be used to make antibodies with specific functions, such as neutralizing antibodies, inhibiting or exciting antibodies, which play important roles in development of therapeutic antibodies and vaccines.