The Preparation Of Monoclonal Antibody Of Major Surface Protein 5 (MSP5) Of Aplasama Marginale Of Dairy Cow And Identification Of It's Epitopes

Anaplasmosis is an obligate intraerythrocytic hemoparasite and tick-borne disease caused by Anaplasma marginale(A.marginale). Anaplasmosis can be found all over the world and mainly cause the death of cattle, sheep, deer and other ruminants. The mortality of cattle can be over 80%. It si one of the most important tick-borne diseases in the animal quarantine. MSP5 which is encoded by a single gene is a highly conserved 19 ku protein in A.marginale, so it is the first selected antigen for the development of the early diagnostic kit. This test prepared A.marginale monoclonal antibody(McAb) with recombinated MSP5 protein(rMSP5), then screened corresponding epitope with the phage display random 12-peptide library. These works lay the foundation for serodiagnosis method and subunit vaccine of Anaplasmosis.The McAbs against MSP5 protein of A.marginale were produced by fusing BALB/c mice myeloma cell line SP2/0 with B lymphoeytes from spleen of BALB/c mice immunized with the purified recombinant MSP5 protein, Two hybridoma cell lines 1D8 and 2F3 that stably produce antibodies against MSP5 protein were identified by ELISA and western blot. The antibody titers of ascites for the McAbs were 106 and 105 respectively. The subtypes of monoclonal antibodies were identified as IgG2b and IgG2a. Additive ELISA indicated that the two monoclonal antibodies were able to recognize identical epitopes of the MSP5 protein of A. marginale. Western blot analysis showed that the McAbs specifically recognized certain antigenic epitopes of MSP5 protein of A. marginael, but not those of other reference antigens.To identify the antigen epitope of A.marginale, McAb 1D8 was purified and screed by the phage display random 12-peptide library kit. The specific phage clones were analyzed for the binding ability with ELISA and competitive inhibition ELISA. And ssDNA of the positive clones were purified for DNA sequencing. After completing sequence analysis , a consensus sequence LING was obtained as linear antigen epitope. The resμLts of the competitive inhibition ELISA demonstrated that the phage clones which had the consensus sequence coμLd compete against recombination MSP5 protein antigen. The specific binding reaction coμLd be found between the McAb 1D8 and the synthetical opypeptide based on LING. A pair of oligonvcleotides was designed based on LING to synthetize epitope gene which had cohesive termini.Then the gene was cloned in the prokaryotic expression pGEX-6-1. It was confirmed that the specific binding reaction coμLd be likewise found between the prokaryocyte expression product of epitope gene and the McAb 1D8 by western blot. The linear antigen epitopes of anti-MSP5 McAb of A. marginale were obtained, which make a good place for investigating the function of the antigen epitope in the diagnosis and vaccine research further.