Preparation And Characterization Of A Single Chain Variable Fragment (scFv) Antibody Against HrpA Protein Of Pseudomonas Syringae Pv. Tomato DC3000

In plant pathogenic bacteria, there is a gene family that regulates the bacteria triggering hypersensitive reaction and pathogenicity (hrp) in plant. In these genes, hrpA encodes a protein named as HrpA. HrpA is a 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 that there is a direct and decisive relationship between existence of the pilus and its causing Hrp protein in host, giving us hints of the possibility to improve plant resistance by blocking assembly of the Hrp pilus through expression of engineered antibodies in plant.In this study, phage display technology was used to screen scFv against HrpA protein of Pseudomonas syringae pv. tomato DC3000. Its HrpA protein was expressed in Escherichia coli, purified and used for screening of positive scFv clones. The scFv DNA was amplified by PCRVith cDNA reverse-transcribed from extracted RNA of hybidoma cells against HrpA protein and ligated into a phagemid vector pCANTAB-5E, and the ligated product was then transformed into E. coli TG1 to yield recombinant phages after infection with helper phage M13KO7. After 5 rounds of panning with HrpA protein, the phage clones displaying scFv fragments of the antibody were selected by ELISA. Two phage clones showing the strongest positive signal in ELISA were obtained to express scFv.In a summary, our results will provide new gene resources for gene engineering for plant resistance and plant antibody.