Preparation And Application Of Specific Endotoxin Removal Adsorbents

Sepsis is a major life-threatening disease caused by endotoxin into the blood,and the detection and removal of endotoxin in various biological products is also a major difficulty.Most of the existing endotoxin removal methods are based on electrostatic and hydrophobic effects.Non-specific adsorption of the existing adsorbents results in low detection accuracy and the application restrictions of endotoxin removal in biological products and clinical blood.Therefore,there is an urgent need for a specific,high affinity,and blood compatible endotoxin binding material.Mannose-binding lectin（MBL）and lipopolysaccharide-binding protein（LBP）are proteins naturally present in the human body that have specific binding capacity for endotoxin.Therefore,in this research,the two proteins were used as research objects,and recombinant proteins were prepared by genetic engineering methods and used for endotoxin detection and removal.The specific research content is as follows:（1）Preparation of MBL and LBP:The plasmid pOptiVEC^TM-TOPO^?-FcMBL,which fused the Fc domain of the antibody and MBL,was constructed by genetic engineering and transformed into human embryonic kidney HEK293F cells with an expression level of FcMBL of 110 mg/m L.FcMBL with a purity of 95%or more was obtained by Protein A affinity chromatography.The LBP gene was separately constructed into Pichia and E.coli expression vectors and failed to achieve high expression in yeast.However,in E.coli Shuffle T7（DE3）,the soluble expression of LBP was approximately 30-50 mg/L,and was purificated by metal ion chelation chromatography with a purity of more than 90%.The ELISA test showed that both of the prepared proteins had the ability to bind endotoxin and the binding capacity of FcMBL was higher.The equilibrium dissociation constant between FcMBL and endotoxin was3.839×10^-7 M measured by BLI method.（2）Establishment of endotoxin detection method:The FcMBL sandwich ELISA method and other two commonly used endotoxin detection methods were optimized and compared.The results showed that the FcMBL-based sandwich ELISA method was suitable for the detection of endotoxin concentrations of 10-250 ug/ml,but the sensitivity was not high enough due to the low binding capacity;under the optimized reaction conditions,the chromogenic substrate hydrazine assay was suitable for 0.005-0.5 EU/m L endotoxin concentration detection;SDS-PAGE with a silver-stained method and a band gray-scale scanning method are suitable for endotoxin detection of 0.03-2 mg/mL.（3）Research on FcMBL adsorbent preparation and evaluation:The FcMBL was immobilized on protein A magnetic agarose microspheres and double epoxy activated sepharose CL-6B to prepare adsorbents.Adsorption kinetics,isothermal adsorption experiments,and adsorption specific experiments were used to conclude that the adsorption capacity of magnetic beads for each 1 mg/m L endotoxin was 0.4 mg/mL,and the agarose gel adsorbent and The adsorption capacity of the positive control polymyxin B adsorbent reached about 3.5 mg/mL,but the adsorption specificity of the FcMBL adsorbent was significantly higher than that of polymyxin B.