Cloning, Prokaryotic Expression Of Three Lipida Synthesis Enzymes And Study Of Casp4Expression Of RAW264.7Cells Stimulated By B.Melitensis Lipopolysaccharide

B-LPS is a major component of the cell wall of Brucella. It mainly consists of three parts:O-antigen, core polysaccharide and lipidA. LipidA is hydrophobic group of B-LPS, and it can make hydrophilic polysaccharide and O-antigen adher to the outer surface, contributing to forming a complete cell wall; Meanwhile, LipidA is also active ingredient of endotoxin and essential for B-LPS structural and functional integrity.Caspase-4is a member of the inflammatory Caspase family and play important roles in the occurrence of inflammation. It is intracellular innate immune receptora of LPS, and has low expression levles in most normal tissues.In the second chapter, the cloning, prokaryotic expression and bioinformatics of three important catalytic enzyme genes, LpxC, LpxK and KdtA, related to the synthesis of B-LPS LipidA were performed. The results showed that we did cloning and prokaryotic expression of thesed three genes sucessfully; By bioinformatic analysis, we gained a preliminary understanding of these three catalyze enzymes' secondary structure and hydrophobic region; Through homologous analysis of the three genes among different brucella species, we found that the three gene sequences are relatively conserved and have high homology among species. Through the study of this part, we gained a better understanding about the process of B-LPS LipidA synthesis, laying a foundation for further study of the function of B-LPS.In the third chapter, we used dose-dependent and time-dependent B-LPS to stimulate RAW264.7cells, then analyzed the changes of Casp4protein expression and IL-1?, TNF-? secretion. The results showed that B-LPS stimulation could up-regulate RAW264.7cells Casp4protein expression and cause the increasing of IL-1? and TNF-? secretion.In the forth chapter, small interfering RNA was used to inhibit Casp4protein expression under B-LPS stimulation, Western blot was performed to detect gene silencing effect, and then ELISA was used to detect the changes of cytokine secretion post-transfection. The results showed that siRNA can inhibit the expression of Casp4protein effectively under B-LPS stimulation. Under the condition of B-LPS stimulation, the knockdown of Casp4with siRNA can make RAW264.7cells to reduce the secretion of IL-1? and TNF-?. These results provid possibility for the study of reducing inflammatory injury through inhibiting upstream inflammatory pathways, and provid an important basis for exploring the pathogenesis of Brucella.