Research On Screening And Function Of Neutralizing Antibody Against VgrG1a Protein Of Pseudomonas Aeruginosa Type Ⅵ Secretion System

Pseudomonas aeruginosa is the main pathogen of nosocomial infection,and the treatment of Pseudomonas aeruginosa infection still requires the joint application of sensitive antibiotics.A new treatment is needed to control the raging of Pseudomonas aeruginosa.This study provides the basis for the treatment of Pseudomonas aeruginosa infection by preparing and screening mouse-derived monoclonal antibodies to the type Ⅵ secretion system（T6SS）VgrG1a protein,with completing humanized transformation of monoclonal antibodies and exploration of efficacy in vitro.This study aimed at the needle protein-VgrG1a of Pseudomonas aeruginosa typeⅥsecretion system（T6SS）.To study the optimal inducing condition of VgrG1a protein,we transformed the designed plasmid coding VgrG1a protein into the competent cell-BL21（DE）p Lys S.Then VgrG1a protein was acted as antigen to immunize BALB/c mice.Hybridoma cells were produced after fusion B cell with P3X63Ag8.653 myeloma cells.After twice screening of 96-well plates and 24-well plates,some monoclonal antibodies with high specificity to VgrG1a were obtained.Next antibodies with the highest affinity were sequenced.After obtaining the sequence information,we designed a kind of plasmid coding 8A4F5 transfected into Expi293 cells to obtain human-mouse chimeric monoclonal antibodies.In addition,Pseudomonas aeruginosa were co-cultured with Klebsiella pneumoniae ATCC700603 and Escherichia coli ATCC25922 in vitro respectively.Thus,a model of inter-bacterial competition was established to explore the role of anti-VgrG1a protein monoclonal antibody in the competition.The optimal induction condition of VgrG1a expression in Escherichia coli was conducted as follows:0.5m M IPTG was added in the logarithmic growth phase and then Escherichia coli were cultured in 16℃shaker for 24 hours.After twice screening of cells in 96-well plates and 24-well plates,five monoclonal antibody-producing cell lines were obtained as follows:6D9E10,11H5G9,6G2D9,8A4F5,2G5F10.Human-mouse chimeric 8A4F5 antibody was expressed in Expi293 cells.According to enzyme-linked immunosorbent assay,it was found that the affinity of human-mouse chimeric 8A4F5antibody to Pseudomonas aeruginosa VgrG1a protein was higher than that of murine8A4F5 antibody.The dissociation constant KD was 6.523×10^-11.In the co-culture of Pseudomonas aeruginosa and Klebsiella pneumoniae,there was T6SS-mediated competitive inhibition,which began in 6～12 hours after co-culture.However,there was no T6SS-mediated competitive inhibition in the co-culture of Pseudomonas aeruginosa and Escherichia coli.The effect of anti-VgrG1a protein monoclonal antibody in inter-bacterial competition was not obvious.In this study,murine 8A4F5 antibody with good specificity against Pseudomonas aeruginosa VgrG1a protein was prepared and screened,and humanized modification was completed.In the future,the role of this antibody against Pseudomonas aeruginosa infection in vivo and in vitro is expected to be further explored..