Purified lipopolysaccharide(LPS)was first obtained from fish-derived Aeromonas hydrophila(A.hydrophila)by hot phenol-water combined with enzymatic hydrolysis.Subsequently,different concentrations of LPS(0,0.2,0.4,0.8,...Purified lipopolysaccharide(LPS)was first obtained from fish-derived Aeromonas hydrophila(A.hydrophila)by hot phenol-water combined with enzymatic hydrolysis.Subsequently,different concentrations of LPS(0,0.2,0.4,0.8,and 1.2 mg/mL)were injected intraperitoneally into Qihe crucian carp(Carassius auratus var.Qihe)with a dose of 3μL/g body weight to evaluated their immunostimulatory activity by detecting the expression of immune-related genes in the spleen,head kidney,and hepatopancreas.The results showed that the average extraction rate of LPS was about 1.063%based on the precipitation weight of wet bacteria,of which the polysaccharide content was 9.39%.The silver staining band of LPS after SDS-PAGE electrophoresis showed a ladder distribution typically characterized by smooth-type LPS.Protein and nucleic acid electrophoresis results showed very low amounts of residual protein and nucleic acid,and high-performance liquid chromatography(HPLC)results showed that the purity of the extracted A.hydrophila LPS was similar to that of high-purity commercial LPS.Pro-inflammatory cytokine genes such as IL-1,IL-8,TNF-α,and IL-6 were more sensitive to LPS stimulation than anti-inflammatory factors such as IL-10 and TGF-β.After 2 h of injection,their expression levels in the spleen,head kidney,and hepatopancreas were significantly increased(P<0.05),with the 0.8 and 1.2 mg/mL injection groups showing the most significant increase.The gene expression of IL-10 and TGF-βshowed slight fluctuations in response to A.hydrophila LPS stimulation(P<0.05).In conclusion,high-purity LPS was extracted from A.hydrophila derived from fish by modified hot phenol-water method,it demonstrating that LPS product could significantly regulate the innate immunity of Qihe crucian carp,with the most effective vaccination dose being 2 mg/kg/BW.These findings provide useful information for future research on endotoxin vaccines.展开更多
基金supported by the General Program of National Natural Science Foundation of China(32373149).
文摘Purified lipopolysaccharide(LPS)was first obtained from fish-derived Aeromonas hydrophila(A.hydrophila)by hot phenol-water combined with enzymatic hydrolysis.Subsequently,different concentrations of LPS(0,0.2,0.4,0.8,and 1.2 mg/mL)were injected intraperitoneally into Qihe crucian carp(Carassius auratus var.Qihe)with a dose of 3μL/g body weight to evaluated their immunostimulatory activity by detecting the expression of immune-related genes in the spleen,head kidney,and hepatopancreas.The results showed that the average extraction rate of LPS was about 1.063%based on the precipitation weight of wet bacteria,of which the polysaccharide content was 9.39%.The silver staining band of LPS after SDS-PAGE electrophoresis showed a ladder distribution typically characterized by smooth-type LPS.Protein and nucleic acid electrophoresis results showed very low amounts of residual protein and nucleic acid,and high-performance liquid chromatography(HPLC)results showed that the purity of the extracted A.hydrophila LPS was similar to that of high-purity commercial LPS.Pro-inflammatory cytokine genes such as IL-1,IL-8,TNF-α,and IL-6 were more sensitive to LPS stimulation than anti-inflammatory factors such as IL-10 and TGF-β.After 2 h of injection,their expression levels in the spleen,head kidney,and hepatopancreas were significantly increased(P<0.05),with the 0.8 and 1.2 mg/mL injection groups showing the most significant increase.The gene expression of IL-10 and TGF-βshowed slight fluctuations in response to A.hydrophila LPS stimulation(P<0.05).In conclusion,high-purity LPS was extracted from A.hydrophila derived from fish by modified hot phenol-water method,it demonstrating that LPS product could significantly regulate the innate immunity of Qihe crucian carp,with the most effective vaccination dose being 2 mg/kg/BW.These findings provide useful information for future research on endotoxin vaccines.
