| Clostridium gas capsule（Clostridium perfringens,C.p erfringene）is made up of Wlechii and Nuttad bodies（1982）separated by corruption in blood vessels,a conditional pathogenic bacteria,the fungus to bacillus,e.coli bacteria of the genus Clostridium,Its colony is translucent and has the characteristics of smooth convex,neat edge,mostly in the form of thick rod,can grow under general conditions,the optimal growth temperature is 37～47℃,can decompose glucose,maltose,produce a lot of carbon dioxide and hydrogen,but not easy to form spores.Spores can be formed in a sugar-free environment with adequate temperature,PH value and carbon source ratio,and spores can survive for 1～4h at 114℃.It is a kind of obligate anaerobe with mild condition and is also a typical toxin pathogen.It is widely found in nature and human living environment.It mainly acts on human digestive system and is common in human and animal intestines,food,soil and feces.Therefore,it can cause human gas gangrene and other diseases and is an important pathogenic microorganism.There are 12 kinds of exotoxins,such asα,β,ε,ι,γ,δ,θ,η,κ,λ,μandυ,among which the first four can cause diarrhea,necrotic enteritis and enterotoxemia in a large number of animals,food poisoning and dysentery in humans.Therefore,according to antitoxin neutralization tests of the four fatal toxins ofα（CPA）,β（CPB）,ε（ETX）,andι（CPI）,they are classified into five categories A,B,C,D,and E.Of these five categories,αtoxins produced by Clostridium percapsulatum type A are the key pathogenic factors for various diseases.The whole human Sc Fv-8B against Clostridium perfringens type Aαtoxin was obtained from the whole human single-chain antibody library Tomlinson I+J by using phage antibody library technology,and the prokaryotic expression vector of Sc Fv-8B gene was constructed,and the expression carrier of Scherichia coli Sc Fv-8B was obtained.In order to increase the stability of single antibody,this research will be to change single antibody,the Sc Fv–8B and different structure constant region important antibodies are linked together,to build a single molecular antibody,screening immune binding activity and obtain optimal single molecule antibody engineering bacterium,ferment cultivation of engineering bacteria,grope for purification process,for single molecule antibody is tasted,The affinity and immune-binding activity of antibodies were verified by in vitro experiments,and the biological activity of antibodies was verified by animal experiments.The main results are as follows:1.Based on the whole human single-chain antibody Sc Fv against CPA screened from the whole human phage antibody library,the ATG-Trx A-Histag-Sumo-（+++++）-Tag expression vector was constructed.Five expression plasmids were synthesized by combining the expression vector with the light chain and heavy chain constant region genome of the single-chain antibody,which were:PET-300-Trx A-Sumo-Scfv-CL-CH2-CH3,PET-300-Trx A-Sumo-Sc Fv-CH2-CH3,PET-300-Trx A-Sumo-Sc Fv-CL-CH2,PET-300-Trx A-Sumo-Sc Fv-Sc Fv-CH2,Pet-300-Trx A-sumo-Sc Fv-CL was transferred into E.coli BL21（DE3）expressing bacteria,respectively,to obtain five monomolecular antibody expressing bacteria.After induced expression by IPTG,EILSA was used to detect the antigen immune binding activity of five monomolecular antibody proteins.Sc Fv-CH2-CH3（Sc Fv-Fc）showed the highest binding activity,so the recombinant antibody protein was used to complete the subsequent experiments.2.The recombinant expression E.coli BL21（DE3）/PET-300-Trx A-Sumo-Sc Fv-Fc was cultured and the fermentation product was purified,and finally Sc Fv-Fc pure product（0.15mg/m L）was obtained.LSPR kinetic analysis showed that,The affinity constant（KD）between Sc Fv-Fc and CPA was 4.89×10-9（M）,indicating that Sc Fv-Fc has a strong affinity for CPA.3.Animal experiments to verify the biological activity of the single molecule antibody.In the in vivo neutralization test,2×LD50 CPA and Sc Fv-Fc were mixed and incubated at different proportions.When CPA and Sc Fv-Fc were mixed at 1:7 and 1:8,almost all the mice were protected.When CPA and Sc Fv-Fc were mixed at 1:6,the mice began to die.The minimum dose of Sc Fv-FC for neutralizing 2×LD50 CPA was 14.21mg/kg.In the challenge protection test,2×Sc Fv-Fc（28.42mg/kg）antibody was injected into the tail vein of mice and challenged at2×LD50 CPA in different time periods.The results showed that the challenge protection rate of the injected antibody could reach 100%within 1 hour.In the challenge treatment experiment,2×LD50 CPA was used to challenge the CPA,and the single molecule antibody was used to treat the CPA at different times.The results showed that the rescue rate reached 90%within 30min and 60%within one hour,indicating that Sc Fv-Fc has a good effect on the rapid treatment of CPA.The half-life of Sc Fv-Fc in vivo was 198min±34min,which was significantly longer than that of Sc Fv（65min±30min）without improvement.This study successfully build A resistance type gas capsule clostridium alpha toxin of recombinant antibody Sc Fv-Fc,single molecule and its property was studied in vivo and in vitro,the results showed that build the single molecule antibodies with high stability,high affinity and long half-life,this study important transformation of protein and to provide experiment basis for safety evaluation... |
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