Construction Of Natural Single Domain Antibody Library Of Bactrian Camel And Preparation Of Heavy Chain Antibody Specific Polyclonal Antibody

In addition to conventional antibody IgG1 in serum of camel, there are two subtypes of heavy chain antibody IgG2 and IgG3. They have the advantages of small molecular weight and strong stability, but the research and application of camel heavy chain antibody was certainly restricted since the market is still lack of commercialization of the camel heavy chain antibody specificity antibodies. It has some application prospect to prepare the polyclonal antibody that specific for camel heavy chain antibody. Camel variable domain of heavy chain antibody (VHH),is the smallest fragment of antibody molecule with the complete function. The antibodies showed a broad application prospect in the fields of basic research and drug development. It offers a great convenience for the preparation of the specific antibodies by displaying the VHH on phage surface, and in vitro screening the polypeptides with desired properties from the colonies contain a large number of variants.In the first part of this study, we combined the advantages of-camel heavy chain antibodies and phage display technology. Total RNA was extracted from peripheral blood lymphocytes of five non-immunized healthy bactrian camels, then reverse transcribed to cDNA,and amplified by PCR to obtain the gene which encoding the camel antibody variable region. By ligation with the phage vector, the recombinant phage was electroporated into competent cells of E.coli TG1 to construct of a natural single-domain antibody libraries of bactrian camel. We identified the library and found library size was 4.4×107, and the transformation positive rate was 75% and with a good diversity.In the second part of the study, the camel plasma was obtained by separat-ing lymphocytes from the camel whole blood with lymphocytes isolation kit, and then the three subtype antibody of camel was separated and purified through Protein G/A Resin column. After three times immunization of rabbit with camel IgG2, the rabbit polyclonal antibodies were prepared in allusion to heavy chain antibody of camels, and its activity was identified. Indirect ELISA results showed the titer of polyclonal antibody was 1:4096. By combining indirect ELISA and Western-blot determine the specificity of the antibody, we got that the polyclonal antibody has no any cross reaction with cattle IgG and camel IgG1, but has reaction with IgG3. In this study, camel heavy chain antibo- dy specific polyclonal antibody was prepared successfully.