| 1. Background and ObjectiveClostridium botulinum is a kind of anaerobic, gram-positive spore forming bacteria, botulinum toxin (botulinum neurotoxin, BoNT) is a secreted by Clostridium botulinum neurotoxin, most have found biological toxins and chemical toxic substances, it can act on the peripheral motor nerve endings, neuromuscular junction, namely the synapse, inhibit the presynaptic release of neurotransmitter acetylcholine, causing muscle relaxant paralysis. Food contamination, the infant intestinal infection and wound infection is the leading cause of human botulinum toxin poisoning. Botulinum toxin consists of A-G seven serotypes, A, B type is the most common type of human botulism, E, F occasionally causes food poisoning epidemic, C, D only cause animal poisoning, G rare. The botulinum toxin molecules have many similar characteristics in structure and function:by the heavy chain (H chain, relative molecular mass of 100KD) and light chain (L chain, relative molecular mass of 50KD), two are connected by 1 two disulfide bonds. Heavy chain C-terminal (Hc), also known as the receptor binding region, which is responsible for binding with target cells; heavy chain N (HN domain) is a transmembrane region, and the light chain with zinc endopeptidase activity. Combined transport and catalysis, the 3 functional areas are linearly arranged, transfer area is located in the middle of Folding spherical structure composed of compact catalytic region by a-helix and β-, transshipment area is mainly composed of a-helix, binding region is composed of two subunit.Clostridium botulinum toxin is a single polypeptide initially produced no activity, relative molecular mass of 150KD, followed by endogenous protease bacterial action itself is decomposed into two chain (H chain and L chain), and the activation of its activity. Single chain neurotoxins into double chain, improve the toxin virulence. But the production of botulinum toxin E of Clostridium botulinum is lack of endogenous protease, and botulinum toxin type E is present in single form, only in host tissues after absorption, is activated in the host protease under single-chain toxin activity to. The effects of the botulinum toxin binding, including location and paralysis in 3 stages of. Once the indirect receptor toxin and neural cell receptor or endocytosis after binding, antibody cannot neutralize the toxin. Binding of the toxin and receptor, endosomal acidic changes, start the toxin transfer area on a conformational change, at the same time the vesicle lumen in the vesicle membrane proton pump ATP enzyme acidification, exposes a hydrophobic region, produce an ion channel in the membrane, L chain through the channel into the cytosol. In cells, three proteins released Ach break down toxins, cause muscle relaxation or spastic paralysis.Botulinum toxin is the most toxic bacterial proteins known human poisoning caused by,0.1～1μgA botulinum toxin A can, or even death.Human botulism are of 4 main types of:① food botulism;② infant botulism; ③ botulinum toxin inhalation poisoning, such as in the laboratory had Botox inhalation poisoning, which is a potential by poisoning the air, there may be used by terrorists or used to manufacture biological warfare agent; the traumatic botulism, similar to tetanus. Botulinum toxin can be a unimolecular termination of a nerve cell function, connected by inhibiting the release of acetylcholine in neuromuscular and effect of poisoning serious death due to respiratory muscle paralysis resulting in respiratory failure. Because of the characteristics of highly toxic and easy production, botulinum toxin type A is classified as a category a bioterrorism agent,was listed as the six most important biological warfare agent. Because of the botulinum toxin from Clostridium botulinum widely exists strong resistance to the external environment and its spores in nature, botulinum toxin poisoning incidents have occurred from time to time, cause serious public health problems.Early in 1817, Justinus Kerner first recognize that some toxins, carrying food, can cause human skeletal muscle and parasympathetic nerve paresis. To 1895,the Van Ermengem in an epidemiological survey of an outbreak of food poisoning, food and human body were detected gram-positive, anaerobic Clostridium botulinum (Clostridium botulinum), and named it "botulinum disease (botulism)". In China, the earliest found botulism is in the founding of the 50’s, in Qapqal Xibe Autonomous County of Xinjiang autonomous region often break out for unknown reasons a called "Qapqal disease" epidemic caused the government’s attention. In 1958, the investigation group composed of Professor Wu Zhaoren to go to the local investigation, according to follow-up, clinical observation and incomplete laboratory test results, the initial diagnosis of the disease is botulism, also suggests that China may have botulism occurred in. Since then, China successfully developed reagents and treatment of botulism diagnosis prevention with antivenom, confirmed the "Qapqal" of type A botulism. In addition, found in other provinces botulism cases have been reported, so far at least has spread to 20 provinces. Xinjiang basically is A type, while other regions except a few of A, E, all of B type. Indicates that China’s botulism type has some regional tendency.In foreign countries, botulinum toxin type A may have been dozens of countries such as the former Soviet Union and Iraq for the preparation of biological weapons, and botulinum toxin can easily be used by terrorists, spread by aerosol botulinum toxin or for the contamination of food and water supplies in the manufacture of biological terrorism (bioterrorism). After the anthrax events and 9.11 events, with the international terrorism is rampant, the human face botulinum toxin poisoning threat degree is increasing. Used BoNT pollution canned orange in early 1984 terrorist organizations, resulting in two U.S. submarines and Bangor base 63 people were poisoned and 50 deaths; in the years 1991-1995 Japanese AUM Shinrikyo (the Japanese cult AumShinrikyo) has tried repeatedly to release of botulinum toxin [11]. In addition, Desert Storm troops to launch (Desert Storm,90/91) 580 soldiers action of toxoid vaccine (PBT) vaccination for the prevention of botulinum toxin, in order to prevent the release of botulinum toxin biological weapons. By 2002 there were 8000 military personnel were inoculated with PBT vaccine [13].In addition, for the maintenance of national biosafety, participate In counterterrorism operations staff also try PBT vaccine [14]. Botulinum toxin as a biological weapon, or as a bioterrorism agent, or occasionally botulism and emergency, its potential harm cannot be estimated. The country must consider the prevention and treatment of botulinum toxin in the formulation of defense strategy. Therefore, research on preventing botulinum toxin has important practical significance and great social value to the national biosafety, public health security and people’s lives and health and safety.2.Research methods2.1. Protective antigen preparation of botulinum toxin type A heavy chain C terminal(1) Prediction of botulinum toxin type A heavy chain C terminal epitopePrediction method of two level structure of this research is:input of botulinum toxin type A column in the database GenBank protein search, select protein containing full gene sequence retrieval, determine the protein sequence of heavy chain in the district as a prediction of the object, using the DNASTAR software, using Gamier program, two secondary structure prediction the protein sequences.Prediction two level structure by using DNAStar Protean software module, with Chou-Fasman crystal structure from the amino acid residues with a predicted two protein structure; the possibility of using Garnier-Robson method to calculate the specific amino acid residues in particular internal structure; prediction of protein by Karplus-Schuhz method and flexible framework. With 7 amino acid residues as a set of programs, prediction of hydrophobic and hydrophilic regions of protein by Kyte-Doolittle method; the possibility to predict specific area located on the protein surface using Emini principle; Jameson-Wolf method application of hydrophilicity, surface characteristics, flexible and two level structure of 4 kinds of joint parameter called antigen index comprehensive prediction of protein, according to the results of the main antigenic domain (Mainan-tigenic domains, MAD).In addition, prediction method of antigenic index can also be used for Wu Yuzhang and other R & D or application server (http://www.expasy. Org/cgi-bin/protscale) to predict the B cell epitopes, according to BoNT/A amino acid residues of hydrophilicity, accessibility, two levels of structure parameters, prediction of antigen epitope formation, followed in the prediction scheme principle is the choice of β angle and beta sheet and random coil is easy to form a regional B cell epitopes, multiple parameter predictionThe method of mutual reference, balance the various forecasting parameters, followed in the prediction scheme is the principle of selection of beta angle and beta sheet and random coil and so easy to form a regional, epitope analysis, inference of botulinum toxin type A heavy chain may antigen epitope.(2)The antigenic polypeptide synthesis of botulinum toxin type A heavy chain C terminalAccording to the prediction results of the botulinum toxin type A heavy chain C terminal epitope, let Shanghai Gil biochemical Corporation synthetic peptides.2. Botulinum toxin type A heavy chain C end peptide antigen monoclonal antibody preparation(1) The immunization of miceFrom 6-8 week old female Balb/c mice immunized with 0.5mg, the first BoNT/ A synthetic antigen with an equal volume of Freund’s adjuvant mixing (lml +1mlFCA), emulsion to drop not after subcutaneous multi-point injection, the injection volume was 200μl/mice. After 2 weeks,0.5mg BoNT/A synthetic antigen with equal volume of Freund’s incomplete adjuvant (lm1+1mlFIA) mixing, emulsifying to drop not after subcutaneous multi-point injection, the injection volume was 200μl/mice. Two weeks later, the mice tail vein blood by centrifugal serum test titer (detected by ELISA). After two weeks,0.5mg BoNT/A synthetic antigen with equal volume of Freund’s incomplete adjuvant (1ml+1mlFIA) mixing, emulsifying to not dripping after intraperitoneal injection in mice, the injection volume was 200μ1/ mice. After two weeks, serum antibody titers detected by indirect ELISA tail by tail vein of mice serum titer, if more than 500000, can be carried out were injected with antigen and PBS (without adjuvant) mixture, three days after the blood test titer after cell fusi(2) The fusion of cellThe culture in NS-1 cells and spleen cells of logarithmic growth lotion appropriately diluted postscript number, two cells were mixed according to the ratio of 1:4,1200r/min centrifugal 12min, go to the supernatant, the same method of cleaning cells 2. With palm centrifuge tube will be at the bottom of the centrifuge tube impact cell mixing. The fusion tube in 40℃ water bath,1ml straw preheat to 40℃ 50%PEG (PH8.0) into the fusion tube, while drops while gently rotating the fusion tube,90s uniform finished. Then add 3ml preheated to complete medium at 37℃,1min uniform finished. Complete medium adding 20ml preheat to 37℃, slowly adding the fusion tube, after the dropper cells by gently pipetting, make the cell mixing (not blow too), the cell suspensions with complete medium volume to 40ml, gently mixing cell. In the shop has 96 holes on the plate feeder cells and add 2 drops/fusion cell suspension, the fusion cell culture plate markers placed at 37℃, 5%CO2 culture box,8 hour after fusion of adding HAT selective medium (HAT final concentration 1 x).(3) Cell screeningThe fusion of the cells in 37 DEG C,5%CO2 culture box. The examination has no pollution, the 3D 1-2 HAT culture medium; general 5-6D visible small cloning; cloning hole radius change of liquid 7d,50%HAT+50%HT for solution, and taking the supernatant for ELISA supernatant titer; 8～10d after cell culture around the hole area to 1/3 when absorb small supernatant for screening, and cultured in HT medium was replaced, Kami Kiyo was positive hole, the higher titer and single cell mass, as the target clone hole. ELISA was used to detect the positive clone free, vigorous growth of holes which are limited dilution cloning of strong positive, cell.(4) CloningClumps of fusion cells positive holes were cloned by limited dilution method. The method is as follows:cloning of Balb/c mice as feeder cells, the positive cells were counted, suspension under inverted microscope, and then use the HT IMDM (containing 15% fetal bovine serum) to adjust the cell number. As the cell count, and then use the HT IMDM (containing 15% fetal bovine serum) to adjust the cell number. Select Al, B1 Weibao hole hole hole, Al:lessons from the positive hole in 100ul cell suspension in A1 hole as conservation hole hole hole; B1:from A1 to learn 10ul cell suspension in B1 hole, mixing, under a microscope using cell counting plate count. Then draw cell suspension in 10ml complete medium from B1 hole, the number of cells in 1-1.5/hole board, each hole 100ul. In 37℃, incubator culture of 5%CO2 cells was observed, the very next day whether there is contamination. The fifth day, every day of the holes in the cell cluster number, and examined the ELISA of culture supernatants, strong positive hole on a single cloning mass, high titer of extensive cultivation. Once again, cloning, and the first method is basically the same, ensure that the 1-1.5 cells/hole.(5) Determination of ascites preparation and titerPositive clone bore down cells by gently pipetting, cell counting plate count, not completely in 0.5ml IMDM medium, a week before injection of paraffin oil or drop the mouse peritoneal phytane.2 days later the mice were observed to have no death or depression symptoms,7 days later, mice abdomen obvious expansion, with 16 needle tube drainage and collected ascites, ascites 10000r/min centrifugal 3min will collect good, Torikami Kiyo.-20℃ frozen ascites. Indirect ELISA assay, cell culture supernatant and ascites were 10 times and 100 times from the beginning of gradient dilution, and NS-1 cell culture supernatants of doing the same dilution was used as positive control.(6) Determination and purification of monoclonal antibody subtypeAscites mAb coating solution was diluted to 1:50000, immunoglobulin subtype identification. With 50-100ul/adding amount of enzyme label plate hole hole,4℃ or 37℃ for 2 hours of adsorption. Discard the hole of the liquid, and PBST washing 3 times, each time 3-5 minutes, pat dry, hard. Each hole with 200ul 5% skim milk powder 7℃ closed 2 hours. Washing 3 times, each hole with 50～100 μ1 detected mAb ascites, and the establishment of nonimmune mouse serum as the negative control and PBS control; 37℃ incubating for 1-2 hours; washing, and pat dry. Two antibody with ELISA (IgGl, IgG2a, IgG2b, IgG3, IgA, IgM), each subtype two holes, each hole 50-100ul,37℃ incubating for 1-2 hours, wash and pat dry, hard. With the substrate solution, A solution:mix B solution=1:1, each hole and substrate is the developer of 50～100ul, at room temperature for 10 minutes. To terminate the reaction of 25ul～50ul 2mol/L H2SO4, on the enzyme-labelling instrument reads the A450 value. Result:with P/N≥2.1, P> N+3D was positive or. If the negative control hole colorless or nearly colorless, positive control hole clear color, can be directly observed with the naked eye. The purified antibody purification by ammonium sulfate saturation method with ascites, computational protein purification rate, protein purity and protein content3. The results1.Prediction of botulinum toxin type A heavy chain C terminal epitopeIn this study, B cell epitope prediction by means of bioinformatics, establishment of epitope prediction can be obtained quickly technology route of specific antibody against epitope peptide based on screening, synthesis of botulinum toxin type A HC end of the B cell epitope peptide. According to the primary structure of botulinum toxin type A heavy chain, using DNASTAR analysis software, combined with the network server, the hydrophilicity, accessibility, flexibility, and the two level structure and other parameters to predict the B cell epitopes. Finally, forecast a two higher polypeptide antigen:PI:(1164-1179AA):KYASGNKDNIVRNNDRP2:(1224-1238AA):KSKNDQGITNKCKMN2. Synthesis of botulinum toxin typeA heavy chain C terminal epitope peptidesSupport Shanghai Gil Biochemical Co., synthesis of botulinum toxin type A heavy chain C end peptide epitopes of P1 and P2. Analysis by chromatography, protein purity was above 95%, the specifications for the 0.5mg/branch.3. Botulinum toxin type A heavy chain C terminal antigen monoclonal antibody preparation and detectionWith botulinum toxin type A synthetic peptide (P1, P2) immunized Balb/C mice, the preparation of monoclonal antibodies,2 strains of hybridoma cells through cell fusion, screening, cloning, extraction of mouse ascites antibody after identification of the protein subunits IgG and IgM, and detecting two strains monoclonal antibody titer in 10-5～10-6 by immune chromatography assay; antibody purity, SDS-page results showed, antibody bands were clear, high purity; identification of specific reaction antibody only reacted with the A botulinum toxin polypeptide antigen fragment, and does not react with staphylococcal enterotoxin, suggesting good antibody specificity.4. DiscussionThis study by means of bioinformatics for epitope prediction, to establish a rapid access to technology route of specific antibody against epitope peptide epitope prediction based on screening, botulinum toxin type A HC end of the B cell epitopes, and synthesis of the polypeptide. According to the primary structure of botulinum toxin type A heavy chain, using DNASTAR analysis software, combined with the network server, the hydrophilicity, accessibility, flexibility, and the two level structure and other parameters to predict the B cell epitopes. Finally, forecast a two higher polypeptide antigen. Support Shanghai Gil Biochemical Co., synthesis of botulinum toxin type A heavy chain C end peptide epitopes of P1 and P2. Analysis by chromatography, the protein purity above 95%, protein high purity, favorable for the later BoNT/A monoclonal antibody preparation. For identification of protein by ELISA method, showed good antigenicity.This polypeptide as antigen was successful preparation of botulinum toxin type A HC end of the B cell epitope of monoclonal antibody and identification. With botulinum toxin type A HC end of the B cell epitope peptide synthesis (P1, P2) immunized Balb/C mice, the preparation of monoclonal antibodies,2 strains of hybridoma cells through cell fusion, screening, cloning, extraction of mouse ascites antibody after identification of the protein subunits IgG and IgM, and measured two monoclonal antibodies the titer is 10"5～10-6; by saturated ammonium sulfate salting-out of ammonium purification method, the purity of antibody detection, the results of SDS-page showed, antibody bands were clear, purity above 70%; specificity antibody only reacted with the identification of botulinum toxin type A recombinant antigen fragment, and does not react with staphylococcal enterotoxin, suggesting specific antibody well. This study provides a new idea for the engineering antibody rapid obtainment of botulinum toxin type A, lay the foundation for the future to obtain antibodies with high affinity. |
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