Preparation Of Monoclonal Antibody Against The Bacterial Protein HrpA

In plant bacterial pathogens, there is a gene family that regulates the bacteria triggering hypersensitive reaction and pathogenicity (hrp) in plant. These genes are conserved and clustered as a pathogenicity island in most cases. When bacteria grow in hrp inducing minimal medium or in planta, one of the structural gene in the gene cluster, hrpA gene, encodes a protein named as HrpA. HrpA is major component of the filamentous surface appendage, Hrp pilus, which plays a very important role in the interaction between bacteria and their host plant. It has been demonstrated there is a direct and decisive relationship between existence of the pilus and its causing hrp in host, giving us hints of the possibility to improve plant resistance by blocking the Hrp pilus assembly through expression of engineered antibodies in planta.To study Hrp pilus function and apply the engineering antibody approach in controlling the disease caused by these bacteria, we employed the sophisticated and time-consuming hybridoma technology. We got two hybridoma cell lines by immunizing mice (BALB/c) with protein purified from engineered E.coli bacteria that confer the hrpA gene of Pseudomonas syringae pv. tomato DC3000, fusing spleen cells with myeloma cells (SP2/0), screening positive candidate with indirect enzyme-linked immunoassay (ELISA), and cloning by dilution for 2 generations. The property of the hybridoma cell lines to secrete expected antibody was confirmed by ELISA and Western blotting analysis. Results showed that the antibody could not merely recognize efficiently and bind specifically to the engineered HrpA that had been used as the antigen, but also the HrpA proteins in pili of the bacteria (Pst DCS 000) induced on the solid hrp inducing medium. However, the monoclonal antibody could not bind engineered HrpA from Erwinia amylovola. The result indicated that the monoclonal antibody we achieved in this study was of high specificity and affinity, which can be used as material in related experiments for better understanding Type III system (TTS) in bacteria, and the hybridoma itself can also be used as the material from which the antibody gene can be cloned in future plant antibody studies.