Preliminary Study On The Production Of IL-1? Induced By Pila From Pseudomonas Aeruginosa

Objective:To study whether the C-terminal part bases of PilA protein of Pseudomonas aeruginosa have an important influence on PilA induces the production of IL-1?,and to provide the basis for future elucidation of the key amino acid sites that PilA affects IL-1?production,so as to ultimately provide an accurate target for the development of new drugs.Methods:1.According to the sequence of pilA gene of PAO1,pil A gene and its124bp deleted at the C-terminus were amplified by PCR,and the PCR amplification fragments were ligated to the plasmid pET-41a?+?.The recombinant plasmids were transformed into E.coli BL21?DE3?and induced by IPTG to express full-length PilA protein?PilA-FL,containing GST-tagged protein?and pilA¹0808 protein which missed 124 bp at the C-terminus(pilA¹⁰⁸,containing GST-tagged proteins).All the protein were purified by GST affinity chromatography to obtain a soluble protein with high purity.2.Pil A-FL,pilA¹⁰⁸and GST-tagged proteins were transfected into mouse macrophage RAW264.7 using liposome transfection method,and a blank group?with no serum and antibiotics in the cell culture medium?and a control group?Lipofectamine?LPS?and liposome?and protein transfection group(GST-tag protein group,Pil A-FL protein group,and pilA¹⁰⁸protein group.In these cell culture medium were added the same culture medium with control group,at the time added0.15?g of the corresponding protein.)During the culture period?6 h,12 h,24 h,48 h,and 72 h?,changes in cell morphology in each group were observed using an inverted microscope.3.At different time points?6h,12h,24h,48h,72h?,the cell culture fluid of each experimental group was taken,and the supernatant was centrifuged to measure the content of IL-1?produced by the El ISA method.Results:1.Successfully constructed two recombinant plasmids,The recombinant plasmids and empty plasmid PET-41a?+?were transformed into BL21?DE3?,After induced expression and purification,a higher purity soluble protein was obtained:GST-tagged protein.,PilA-FL protein and PilA¹0808 protein.2.With the changes of time?6h,12h,24h,48h,72h?,the morphological changed of macrophages in the blank group was not obvious,but the cells in the control group and protein transfection group all changed significantly.In the control group and the protein transfection group,compared with the blank group at the same time point,the cells began to have inflammatory changes at the 6th hour,vacuoles and toxic particles appeared in the cytoplasm,and the inflammatory changes of the cells became more and more serious as the time went by.The cytoplasmic vacuoles and toxic particles continuously increased,and they were most typical at 48 h.At 72 h,the cells began to degenerate.3.At 6h,12h,24h,48h,and 72h,IL-1?content in the control group and protein transfection group was significantly higher than that in the blank group?P<0.05?;IL-1?in the protein transfection group The content was significantly higher than that in the control group?P<0.05?;the IL-1?content in the PilA-FL protein group and PilA¹0808 protein group was significantly higher than that in the GST-tagged protein group,and the difference was statistically significant?P<0.05?.There was no significant difference in IL-1?content between PilA-FL protein group and PilA¹⁰⁸protein group?P>0.05?.Conclusion:1.When LPS was used to stimulate macrophages for 6h,the cellular inflammation model was successfully constructed;2.The 124bp deletion of the C-terminus of PilA protein may not have an important effect on IL-1?production by mouse macrophage RAW264.7 induced by PilA protein.3.The key amino acid site for IL-1?production by P.aeruginosa pilus may not be among the 124 bases at the C-terminus of the PilA protein gene.