| Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen among the most important superbugs,which is highly resistant to most of the clinically used antibiotics.The resistance mechanism of Pseudomonas aeruginosa is complicated.For example,antibiotics are difficult to enter the biofilm after biofilm formation,which makes the drug resistance enhanced.Through the type III secretion system,it can invade mammal cells and become intracellular bacteria which makes it difficult for antibiotics to enter the cells and thus is difficult to be killed.Due to the inherent resistance of Pseudomonas aeruginosa in the treatment of antibiotics,it is increasingly difficult to control the infection of Pseudomonas aeruginosa.Therefore,novel treatment strategies are urgently needed.Antibody drugs have the advantages of specificity and safety,and research on the antibacterial antibodies have attracted more and more attention these years.In this study,two single-chain variable region(ScFv-Fc)antibodies were prepared: MFa targeting PcrV protein and MFb targeting Alginage.Then the affinity activities of the antibodies were analyzed to study their in vitro activities against Pseudomonas aeruginosa,especially their effects on Pseudomonas aeruginosa biofilm formation,adhesion to cells,and invading cells.The research provided a basis for further development of novel antibody drugs against Pseudomonas aeruginosa infection.In this study,the eukaryotic expression plasmids were constructed first,which were then transiently transfected into HEK293 E cells by polyethyleneimine(PEI)and antibodies got expressed.The expressed products were purified by HiTrap MabSelect SuRe column,and the obtained antibodies were examined by SDS-PAGE for purity analysis.The antibodies concentrations were determined by BCA assay.In order to study the affinity of the antibodies with their antigens,PcrV and Alginate were expressed or synthesized.The PcrV sequence was inserted into pET28 a,and the recombinant plasmids were chemically transformed into E.coli BL21(DE3).Recombinant PcrV protein was induced to express at low temperature at 15 °C by 0.5 mM IPTG.Affinity purification was performed by His-Tag tag fused with PcrV,and the quality of the purified PcrV were analyzed by SDS-PAGE,BCA,and HPLC;Alginate was coupled with BSA by EDC to develop Western blot and ELISA assays for MFb characterization.The affinity of the antibodies to their respective antigens were analyzed by Western blot,ELISA,and surface plasmon resonance(SPR).We further studied the activity of the prepared antibodies MFa and MFb against Pseudomonas aeruginosa in vitro,mainly in the following aspects: the effect of MFa and MFb antibodies on biofilm formation,biofilm disruption,adhering to HeLa cells,invasion of HeLa cells,and the phagocytosis of Pseudomonas aeruginosa by macrophages.The main results obtained in this study was as following:Firstly,the antibodies MFa and MFb against Pseudomonas aeruginosa PcrV and Alginate were successfully prepared.Affinity experiments showed that the KD of MFa binding to PcrV antigen was 492.2 nM,and the KD of MFb binding to Alginate was 8.308 nM.Secondly,the in vitro biological activity of the antibodies indicated that both MFa and MFb have reduced the formation of Pseudomonas aeruginosa biofilm,the adhesion of Pseudomonas aeruginosa to HeLa cells,and the invasion to HeLa cells.Either MFa or MFb treatment can increase the phagocytosis of Pseudomonas aeruginosa by macrophages.In summary,in this study we successfully prepared two scFv-Fc type antibodies MFa and MFb,targeting PcrV and Alginante respectively.The functions analysis showed that both antibodies can inhibit biofilm formation of Pseudomonas aeruginosa to a certain extent,reduce the formation of intracellular parasites,enhance the phagocytosis of macrophages.The results suggested that the two antibodies were of potential utility for Pseudomonas aeruginosa infection treatment and were promising to be further developed for novel therapeutic strategies against Pseudomonas aeruginosa. |
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