Expression And Purification Of Clostridium Perfringens Alpha-Toxin And Screening Human ScFv Antibody

Clostridium perfringens alpha-toxin(CPA) is the main pathogenic exotoxin causing human gas gangrene and food poisoning,widely exists in the nature. In recent years, there has been an increasing number of food poisoning caused by Clostridium perfringens and its main causative factor is CPA,so people began to research the properties of CPA,including the structure of the CPA,N-terminal domain and C-terminal domain function and gene fusion expression, but there is no in-depth study for acute treatment of CPA after food poisoning and therapeutic antibodies.This study using genetic engineering,both ends of the whole gene sequence of cpa insertion Nde I and EcoR I restriction sites.Then cpa was inserted into the pET-28 a expression vector. The correct clone vector pET-28a-CPA was transformed into E. coli competent cells BL21(DE3). Conditions for the expression were optimized to obtain the best purification, exploration conditions of purification process, the finally purity of 95.53%. With CPA as antigen by Tomlinson I+J human scFv library and screening of anti CPA-scFv,it was induced to expression and purification determination of antibody binding activity, biological activity and affinity constant.Restriction analysis and sequencing proved that the correct ORF of CPA gene was cloned into vector pET-28 a, CPA molecular weight is about 43000 Da in sodium dodecyl sulfate–poly-acrylamide gel electrophoresis. Optimize the expression condition and purification technology, CPA is mainly expressed as soluble form. The optimal medium,temperature for induction and time for induction were TB culture medium,37 and 3 h respectively.The CPA ℃was purified using 30%-50% saturation of ammonium sulfate precipitation, Butyl Sepharose 4 FF, Cu2+-chelating chromatography,anion exchange chromatography(DEAE) and Cu2+-chelating chromatography. The purity,concentration and recovery rate of purified CPA were 95.53%,0.663 mg/mL and 5 mg/g wet bacteria respectively. Through four rounds of screening, obtained two positive clone strains(scFv-7D and scFv-8B), after DNA sequencing and Blast database analysis the strains were human single chain Fv(scFv) antibody.The scFv was purified using 60% saturation of ammonium sulfate precipitation and rProtein-A affinity chromatography. The purity of purified scFv was more than 90%. In vitro we measured its activity by determining its effect on egg yolk lipoproteins, results showed that scFv-8B and scFv-7D have good inhibition effect on the activity of CPA. According to the results of ELISA, scFv immune binding activity with increased concentration increased, the scFv-8B immune activity is higher than the scFv-7D.The affinity constants between scFv-8B and scFv-7D were measured by non-competitive enzyme immunoassay,which are(3.45±0.58)×108 L/mol and(2.01±0.78)×108 L/mol respectively.In this study, We have shown that the production of large amounts of soluble, easy operation and functional proteins by using the pET-28 a vector in E.coli. Not only for protein purification has opened up a new way, but also to further research CPA pathogenic mechanisms and biological characteristics of the food poisoning has great significance. While successfully screened human anti CPA-scFv,they have high biological activity and immune binding activity, but also conducive to the prevention of food poisoning and CPA treatment, after application to avoid the body to produce human anti-mouse antibody response, decrease the adverse reaction, improve safety and efficacy, provide a new method for the treatment of bacterial food poisoning.