| Botulinum neurotoxins(Bo NT) is a kind of potent toxin proteins, produced by anaerobic clostridium botulinum, which is the most poisonous toxin in the world and it received much attention from investigators in many countries not only because it can cause poisoning among the people, but also can be abused as terrorist agents and biological warfare agents.Botulinum neurotoxins is connected by heavy chain(relative molecular mass of 100000)and light chain(relative molecular mass of 50000) with disulfide bond. Light chain(Bo NT/AL) is the toxic component of botulinum neurotoxins, which acts as zinc metallopeptidases. The heavy chain is divided into two domains, N-terminal domain(Bo NT/AHn) plays an important role in the Internalization process by forming an ion channel in the cell membrane. C-terminal domain(Bo NT/AHc) is divided into two domains-AHCC and AHCN. The amino acid of AHCC is more variable than AHCN, accordingly, the domain of AHCC is likely to cause the specificity of the serotypes. Bo NT/AHc is both the acceptor binding region and the determining region of neutrality antigen, which possesses full protective effect. Therefore, the epitope on Bo NT/AHc provides proof for the screening of neutralizing antibody.It is reported that there are 3 acceptors against Bo NT/A, they are respectively gangliosie(GT1b) 、 synaptic vesicle protein(SV2) and fibroblast growth gactor receptor 3(FGFR3). SV2 can be divided into 3 isotypes of SV2A/SV2B/SV2 C, and the bonding force of SV2 C and Bo NT/A is the strongest, therefore, SV2 C can be used to detect the bonding force between acceptor and mutant library. GT1 b binds with Y1098、E1184、F1233、H1234、W1247、Y1248、S1256、R1257 of Bo NT/A. SV2 binds with S1123、M1125、T1126、T1127、Y1130、R1137,R1275、R1160、V1161、Y1162、Y1172、R1173、L1174. The binding sites of FGFR3 is not yet clearly. GT1b、SV2、FGFR3 reform one kind of acceptor complex formation, which could contribute Bo NT/A enter the inside of the cell. Moreover, it is possibly because that the binding of neutralizing antibodies and toxin enclose the binding sites of acceptor and Bo NT/AHc, which could be the mechanism to explan the neutralization of antibody. Therefore, identifying the binding sites of FGFR3 with Bo NT/AHc could contribute to identify the interaction between three acceptors and neutralization epitope of Bo NT/A.No small-molecule drugs exist for the treatment of Bo NT intoxication up to now. Equine antitoxin is a commonly used drug, however, this product exists lots of disadvantages, which is potent serum sickness, anaphylactic shock, high cost and a long period. Therefore, application of this treatment is limited. In recent years, with the fast development of genetic engineering, genetic engineering neutralization antibody appears great potential for replacing equine antitoxin. Peter Amersdorfe et al construced exempt bacteriophage single-chain antibody library against Bo NT/AHc by immunizing mice and gathering SPC,screened 28 antibody in high specificity, which covered 4 non-overlapping epitopes against Bo NT/AHc, afterwards, 28 single-chain antibody against Bo NT/A were characterized and single-chain antibody C25/S25/3D12 were obtained,which possessed high neutralizing activity in vitro. In addition, the epitopes of C25/S25/3D12 were identified respectively,the neutralization epitope of C25 is: N899、L900、E901、F934、R1042、D1043、T1044,H1045、Y1047;the neutralization epitope of S25 is: N899、L900、E901、F934、R1042、D1043、T1044;the neutralization epitope of 3D12 is G1110,I1111,R1112. Nowakowski et al expressed neutralizing antibodies C25/S25/3D12 in cell CHODG44 and detected the neutralizing activity in vivo based on the research results of Peter Amersdorfer. The result showed that the antibodies could protect the mouse from the challenge of 450,000 LD50 of Bo NT/A when used combined, besides, the neutralization effect of optional two antibodies is far less than three.It is likely that toxin protein possesses multiple receptor binding domains even single antibody used couldn’t enclosed these domains. Except this, XOMA company in America screened 3 antibodies against Bo NT/A including NX01/NX02/NX11, whose titer is 500 times of antisera, however, the issue is not only unpublished sequence but also private intellectual property. The laboratory of Zhiwei Sun in our institute obtained 3 monoclonal antibodies C10、1B6、2G4 against different epitopes of Bo NT/AHc from high-capacity complete synthesis bacteriophage antibody library, however, its potency remains to be optimized. It is emphasized that the researches above were started with antibody screening and ended up with the research of neutralization epitope. In contrast, this study will combine epitope analysis and antibody screening,in addition, antibody screening is guided by epitope analysis, from systematic study of the epitope to the research of neutralizing antibodies.This study started with monoclonal antibody preparing and screening,combined toxin epitope forecasting and mutant construction to definite the epitope against monoclonal antibody,in addition, neutralization titer of each antibody group against Bo NT/A was detected according to epitope group and neutralization experiment to screen the most effctive group,which not only processes the best neutralization activity but also includes the least antibody number.In this study, mutant library were forecasted and constructed by Discovery Studio according to the crystal structure of Bo NT/AHc, meanwhile, monoclonal antibodys against Bo NT/AHc were prepared, mutant library was detected with monoclonal antibody to identify the epitope of Bo NT/A preliminarily by ELISA.117 potential epitopes were screened,which referred key sites in paper and previous research in laboratory about Bo NT/A except for Discovery Studio. Every mutant was designed embodying 3-5 amino acids and 30 mutants against Bo NT/AH(p TIG-AHT1~30) were obtained by fast mutagenesis. Besides, 22 mutants against Bo NT/AHc(p TIG-AHc T1~22)were also obtained by use of polymerase chain reaction which setted mutants against Bo NT/AH as template. Afterwards, induced bacterias of 30 mutants against Bo NT/AH、purified proteins of 22 mutants against Bo NT/AHc and acceptors of FGFR3 and SV2 C were detected with ML01/ML02 by ELISA. It was because that binding site of acceptor SV2 C was already known, analysising the difference between mutated amino acid sequences of binding capacity decreased mutants against Bo NT/AHc and definite binding sites against SV2 C could test reliability of this detection device.The result showed that mutated amino acid sequences of binding capacity decreased mutants covered definite binding sites of SV2 C against Bo NT/A, which proved ELISA is effective and reliable as protein interaction detection device. In addition, potential antigen epitopes were screened preliminarily, streamlining and clear of the binding sites need for further experimental study like animal activity experiment and other detection device of protein interaction.Above all, this study constructed 30 mutants against Bo NT/AH and 22 mutants against Bo NT/AHc, besides, 22 mutants against Bo NT/AHc were complished induction,expression and purification, the purity quotients of the protein were all beyond 85%; 6 monoclonal antibodies against Bo NT/AHc were prepared and characterized,named as ML01、ML02、1A4、3H3、3H7、5H8 respectively;potential antigen epitopes were screened preliminarily: 917'921 FNLES、1064'1066 HRY、1127'1131 NVGIR、1163'1167 KKYAS、1240'1242 QDN、1179'1181 RVY、1255'1257 FNN、958'961 LNNE。... |
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