| Tetanus toxin(TeNT)is a protein-based neurotoxin produced by anaerobic clostridium tetani under anaerobic conditions.It can cause human death within about 100 ng,ranking second only to botulinum toxin in toxicity.TeNT poisoning can lead to continuous excitability of human motor neurons,resulting in myotonic contraction.At present,there are no effective chemicals for the treatment of TeNT poisoning,and horse serum antitoxin and human immunoglobulin are often used for treatment in clinic.However,equine serum antitoxin may cause allergic reaction clinically,and the source of human immunoglobulin preparation is relatively difficult,leading to great limitation in application.Horse serum antitoxin and human immunoglobulin are essentially polyclonal antibodies,and both of them have the common problems of polyclonal antibodies,such as poor specificity,large difference between batches,and easy to cause side effects.Better drugs are urgently needed for the treatment of TeNT poisoning.At present,monoclonal antibody drugs are widely used for the treatment of many diseases due to their strong specificity and easy source for polyclonal antibodies.However,there is little research on the monoclonal antibody against TeNT,and no neutralizing antibody has been applied clinically so far.Therefore,it is of great significance to screen the neutralizing antibody against TeNT.In this study,tetanus toxoid and TeNT receptor binding domainTeNT/Hc segment which can stimulate the body to produce antibodies were used as antigens for mouse immunization.The mouse-derived neutralizing monoclonal antibodies with strong specificity and high affinity are screened,evaluated and identified by the hybridoma technology.First,the entire TeNT gene sequence was synthesized and the fragments of TeNT/L,TeNT/LHn,and TeNT/Hc were amplified by PCR to construct recombinant plasmids pNZ8048-TeNT,pNZ8048-TeNT/L,pNZ8048-TeNT/LHn,and pNZ8048-TeNT/Hc,which were introduced into Lactococcus lactis NZ9000 by electro-transformation.The recombinant proteins TeNT,TeNT/L,TeNT/LHn,and TeNT/Hc were induced and expressed at 30℃ under the induction condition of Nisin.The recombinant protein was purified using a His Trap HP affinity column and a PD 10 desalination column,both of which were greater than 95.0%pure by Gel-pro32 analysis.The proteins were identified by Western Blot and all showed specific binding to anti-tetanus toxin antibodies.The virulence of TeNT protein was determined in the mouse anti-virus experiment,and the virulence was 8×105 LD50/mg.The experimental results showed that the recombinant protein TeNT could be used as a toxin in subsequent antibody neutralization evaluation.The recombinant protein TeNT/L,TeNT/LHn and TeNT/Hc can be used as immune antigen required for subsequent hybridoma cell screening,screening antigen and for subsequent epitope analysis experiments.The female BALB/c mice were immunized with tetanus toxoid and the tail blood serum titers of the immunized mice were determined by indirect ELISA.The spleen cells of No.3 mouse with higher tail blood titer were cell-fused with Sp2/0 myeloma cells by hybridoma technology,and the cell fusion rate was 95.31%.After the fusion cells were screened by HAT selective medium,the positive hybridoma cells were screened by indirect ELISA method,and the cell positive rate was 12.29%.After clonal culture of the positive cells,six positive hybridoma cell lines(1A7,2C7,3A7,3H4,4C1 and 4E12)stably secreting antibodies were obtained.The antibody subtypes were all of IgG1/κ type after identification,and all of them were the common subtypes of murine antibodies.The ascites was prepared by allogeneic ascites induction method after the cell strain was expanded and cultured.The ascites was purified by two steps of Protein G affinity chromatography column and PD 10 desalination column to obtain mouse-derived monoclonal antibodies with the purity greater than 95.0%.The binding domain and epitope type of the antibodies were identified by indirect ELISA and Western Blotting.The results showed that all five antibodies specifically bound to the antigen TeNT/Hc by linear epitopes.Only 3A7 antibody specifically bound to the antigen TeNT/L as a linear epitope.The affinity constants of the antibodies against the specifically bound antigens were measured by non-competitive ELISA.The results showed that the affinity constants of the six antibodies against the antigens reached nmol/L level,indicating that the antibodies had high affinity with the antigens.Among them,the affinity of 4E12 antibody has reached pmol/L level,indicating that it has high affinity for antigen.The results of neutralization titer in mice showed that all six antibodies could delay the death of animals.The antibodies 1A7,3A7,3H4,and 4E12 all showed partial protection.The 4C1 antibody was fully protective with a sum titer of 50 LDso/mg.Based on the results of the neutralization titer,the further protective dose for neutralization of 4C1 antibody was 200μg,with a window of 1 h.Female BALB/c mice were immunized with the recombinant protein TeNT/Hc,and the serum titer of tail blood was determined by indirect ELISA.The No.2 mouse with higher titer of tail blood was taken for hybridoma cell fusion,and the cell fusion rate was 86.84%.The positive rate of hybridoma cells screened by indirect ELISA was 7.64%.After clonal culture of the positive cells,seven hybridoma cell lines(1A1,1H7,2D7,4G2,7H12,8D2,8F1)stably secreting antibodies were obtained.Ascites was prepared by allogeneic ascites induction method and murine monoclonal antibodies were prepared by two-step purification method.The purity of antibodies analyzed by Gel-pro32 was greater than 95.0%.The antibody subtypes were all of IgG1/K type after identification,and all of them were the common subtypes of murine antibodies.The indirect ELISA and Western Blotting assays showed that the seven antibodies all specifically combined with the antigen TeNT/Hc by linear epitope.The affinity constants of the seven antibodies against the antigen TeNT/Hc were determined by non-competitive ELISA.The results showed that the affinity constants of the seven antibodies had reached the nmol/L level,indicating that the antibodies had high affinity with the antigen.The results of neutralization titer in mice showed that antibodies 1A1,2D7,7H12,and 8D2 could prolong the survival time of animals,and antibodies 1H7,4G2,and 8F1 had partial protection at higher doses.The neutralizing 4C1 antibody was HRP labeled.The epitopes of the 13 obtained antibodies were competitively grouped by competitive ELISA.The results showed that only 1H7 and 7H12 antibodies had the same epitope as 4C1 antibody,and the other antibodies had different epitopes.The neutralization titer of 4C1 antibody combined with other antibodies with different epitopes was determined by mouse neutralization test.The results showed that the neutralization titer was significantly increased when the antibodies were combined,and the effect was most significant when 3A7 was combined with 4C1.The 10 LD50 TeNT could be completely neutralized when the injection dose of each antibody was 10μg/animal.The combination of 3A7 and 4C1 with 4E12,which had the highest affinity,was continued.The results of mouse neutralization test showed that the titer was also improved after the combination.When the injection dose of each antibody was 6 μg/mouse,complete neutralization of the toxin was achieved.In summary,in this study,the recombinant proteins were expressed by Lactococcus lactis expression system.The purity of the purified proteins was greater than 95%,and the recombinant proteins had good antigenicity.Thirteen monoclonal antibodies against TeNT were screened by hybridoma technique.The affinity constants of the antibodies all reached the nmol/L level,and the affinity of 4E12 antibody reached the pmol/L level.The results of mouse neutralization test showed that all the 13 antibodies had different neutralization protection effects.Among them,the neutralization titer of 4C1 antibody was higher,and the combination of two or three antibodies with other 4C1 antibodies could increase the neutralization titer by 20～30 times.The monoclonal antibody obtained in this study can lay a foundation for the subsequent research and development of therapeutic drugs for tetanus toxin poisoning and the establishment of tetanus toxin test technology. |
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