Development And Characterization Of Monoclonal Antibodies Against Tetanus Toxin Fragment C

Tetanus toxin, one of the most potent toxins known, is a highly potent neurotoxin produced by the anaerobic bacterium Clostridium tetani. According to the function in vivo, the toxin can be separated into A,B and C three fragments. The fragment C (TetC) is atoxic and retains the ganglioside-binding activity of intact tetanus toxin. It has been demonstrated that the immunizing potency of TetC was similar to that recorded for tetanus toxin and monoclonal antibodies (McAbs) obtained from TetC immunized mice had the ability of neutralizing tetanus toxin. TetC was protective antigen of tetanus toxin and the requirement for TetC binding to neurons through gangliosides as a first step in intoxication has been confirmed. In this study, recombinant bacteria expressing TetC gene of Clostridium tetani was constituted; the expressed TetC protein bearing its immunogenic characteristic was acquired and hybridoma cell lines secreting McAbs against TetC were generated. All these results would lay the preliminary foundation for the detection of the biological characteristic of TetC, the relationship between tetanus toxin structure and function, and developing the method in the rapid detection of the titers of tetanus antitoxin in human sera.1. Cloning and expression of tetanus toxin fragment C in E.coliThe TetC gene was amplified from the genomic DNA of Clostridium tetani strain 64008 by PCR and inserted into the pMD19-T vector. After sequencing confirmation, the gene was then inserted into the prokaryotic expression vector pGEX-6p-1 and recombinant bacteria BL21(pGEX-6p-1-TetC) was developed. After IPTG induction, fusion protein was purified by Redipack GST purification module. The results of SDS-PAGE verified that the size of fusion protein was consistent with prediction. Western-blot analysis further indicated this expressed product had the immunogenicity of tetanus toxin fragment C.2. Development and characterization of monoclonal antibodies against tetanus toxin fragment CTo prepare monoclonal antibodies against TetC protein, purified protein His-TetC, which is the product of recombinant bacteria Rosetta(DE3)(pET-TetC), was used as immunogen to immunize subcutaneously or intraperitoneally 8-week-old female BALB/c mice. The immune dose was 100μg of His-TetC protein emulsified in complete Freund's adjuvant for the first immunization and in incomplete Freund's adjuvant for the next injection with 2-week intervals. After the titer in sera by ELISA exceeded 1:10000, an intravenous dose of purified protein was administrated. After 3 days, splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. Purified GST-TetC protein was used as detecting antigen, and the supernatant of hybridoma clones were screened by indirect ELISA. Five hybridoma cell lines secreting McAbs against TetC, named 1E5,5G10,6B9,7D6,7F5 were obtained. The immunoglobulines subclasses of these McAbs were IgG₁. The ELISA titers of 1E5 McAb ascites was 3×2¹⁰, the others were 1×10⁵. Western-blot analysis confirmed that five McAbs could only react with His-TetC protein, but not react with Rosetta(DE3)(pET) or BL21(6p-1), which were used as control. These results suggested that McAbs 1E5,5G10,6B9,7D6,7F5 against TetC have been successfully developed.