Tetanus subtoxin and preparation method thereof

Abstract

The invention relates to tetanus subtoxin and a preparation method thereof. The preparation method specifically comprises the following steps: providing or constructing a tetanus toxin Hc fragment mutant TeNT-Hc strain; inoculating the TeNT-Hc strain into a seed culture medium according to an inoculum size of 0.1%-0.5%, and carrying out shake culture until the OD600 value reaches 0.8-1.5 to obtain a first-stage seed culture solution; further increasing the number of strains on the basis of the first-stage seeds to obtain a second-stage seed culture solution; performing fermentation; re-suspending, crushing and centrifuging the bacteria, collecting supernate, carrying out Ni column affinity chromatography on the supernate, washing impurities and eluting, and collecting eluent; and carrying out second-step purification on the eluent through isoelectric precipitation or Q anion exchange or SP ion exchange, and collecting the supernate or the eluent to obtain the tetanus subtoxin. The purity of 95% or above is achieved through two-step purification, and meanwhile the expression quantity of the target protein is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, in particular to a tetanus toxin and a preparation method thereof. Background Art

[0002] Tetanus is a serious disease caused by infection with Clostridium tetani. Clostridium tetani reproduces in hypoxic conditions and produces tetanus neurotoxin. This toxin enters the nervous system through the bloodstream and acts on the central nervous system, inhibiting the release of neurotransmitters and disrupting communication between nerves and muscles, leading to severe respiratory and hemodynamic disorders. Tetanus toxin is extremely deadly; approximately 100 nanograms can be fatal. Approximately one million cases of tetanus occur worldwide each year, with a mortality rate as high as 50%. In developing countries, the mortality rate for neonatal tetanus is as high as 90%. Therefore, the development of effective and safe drugs for the prevention and treatment of tetanus is a major research priority worldwide.

[0003] Tetanus toxin is cleaved by proteases into a light chain and a heavy chain linked by disulfide bonds. Its functions are divided into three parts: A, B, and C (each with a molecular weight of 50 kD), which respectively play binding, transduction, and paralysis roles, thereby inhibiting neurotransmitter release. The native C fragment (Hc), the C-terminus of the heavy chain, retains many of the properties of the intact toxin that bind to gangliosides. Its immunogenicity is comparable to that of the toxin, but its allergenicity is lower, making it suitable for the preparation of subunit vaccines.

[0004] Many studies at home and abroad have improved the expression of the tetanus Hc segment, and soluble expression products of recombinant Hc proteins have been obtained using Escherichia coli. For example, patent application number 200910135972 discloses recombinant bacteria and recombinant expression methods for in vitro expression of tetanus toxin subunit vaccine Hc. However, the expression level is low, with the soluble target protein expression level being only 300 mg / L. Patent application number 201611256457 discloses a method for increasing expression through high-density fermentation, with the soluble target protein expression level reaching 800 mg / L. However, the purification process is relatively complex, using three column chromatography steps, in which the supernatant is passed through a QFF column for anion exchange, a phenyl hydrophobic column for hydrophobic chromatography, and then an SP column for cation exchange.

[0005] Existing studies have successfully expressed recombinant Hc protein using Escherichia coli, but there are problems such as low expression level or complex purification process.

[0006] Therefore, the existing technology needs to be improved. Summary of the Invention

[0007] In the prior art, recombinant Hc protein has been successfully expressed using Escherichia coli, but there are problems such as low expression level or complex purification process. Therefore, the present invention provides a tetanus toxin and a preparation method thereof to solve the above problems.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing tetanus toxin, which comprises the following specific steps: S1. Provide or construct a tetanus toxin Hc fragment mutant TeNT-Hc strain; S2. Preparation of primary seeds: Sterilize the seed culture medium in a shake flask, cool to room temperature, inoculate the TeNT-Hc strain into the seed culture medium at a rate of 0.1% to 0.5%, and culture in a shaker at 37°C for 3 to 8 hours until the OD 600 When the value reaches 0.8-1.5, the first-level seed culture solution is obtained; S3. Preparation of secondary seeds: further amplifying the number of strains based on the primary seeds to obtain secondary seed culture fluid; S4, fermentation: the secondary seed culture solution is inoculated into the fermentation tank at a rate of 1% to 5%, and a feed medium is added for feeding. When the bacterial density of the bacteria reaches 30 to 40, IPTG is added to induce expression. When the OD value reaches 70 or above, the fermentation tank is removed from the fermentation tank, the fermentation solution is collected and centrifuged to collect the bacteria; S5, first step of purification: resuspending the bacteria, breaking and centrifuging, collecting the supernatant, passing the supernatant through Ni column affinity chromatography, washing and eluting the impurities, and collecting the eluate; S6. Second step purification: the eluate is subjected to second step purification by isoelectric precipitation or Q anion exchange or SP ion exchange, and the supernatant or eluate is collected to obtain the tetanus subtoxin.

[0009] In one implementation, in S1, the TeNT-Hc strain is an engineered bacterium containing the tetanus toxin receptor binding region Hc nucleotide sequence.

[0010] In one implementation, in S2, the seed culture medium includes 5 g / L yeast powder, 10 g / L peptone, and 10 g / L NaCl.

[0011] In one implementation, in S3, further expanding the number of strains specifically includes: placing the seed culture medium into a shake flask for sterilization, cooling it to room temperature, inoculating the first-level seed culture solution into the seed culture medium at a 0.1% to 0.5% inoculum, and placing it in a shaker for constant temperature shaking and culturing at 37°C for 3 to 8 hours until the OD 600 When the value reaches 0.8~1.5, the secondary seed culture solution is obtained.

[0012] In one implementation, S4 specifically includes: S41. Provide a fermentation tank, adjust the volume according to the fermentation medium formula, and sterilize at 110-130°C for 15-25 minutes; S42, the secondary seeds are inoculated into the fermentation tank at an inoculum rate of 1% to 5%, the temperature is controlled at 37°C, the pH is 7.0, and the dissolved oxygen is ≥30%. After the dissolved oxygen rapidly rises to above 60%, feed medium is added for feeding; wherein, the initial feeding rate is 15 g / (L*h), and the dissolved oxygen is controlled to be ≥30% after feeding; S43. When the bacterial density reaches 30-40, start cooling and set the temperature to 16-25°C. S44, add 0.1mM~1mM IPTG for induction, and induce for 8~12h; S45, when the OD value reaches above 70, the tank is unloaded and the fermentation liquid is collected; S46. Centrifuge the fermentation broth and collect the centrifuged bacteria.

[0013] In one implementation, in S41, the fermentation medium composition is: yeast extract 5 g / L, ammonium sulfate 5 g / L, glucose 10 g / L, dipotassium hydrogen phosphate 3.8 g / L, anhydrous potassium dihydrogen phosphate 3.5 g / L, magnesium sulfate heptahydrate 1 g / L, trace elements 1 g / L, calcium chloride 0.075 g / L, and VB1 0.0022 g / L.

[0014] In one implementation, in S4, the feed medium includes 500 g / L of glucose, 20 g / L of magnesium sulfate, and 1 g / L of trace elements.

[0015] In one implementation, the trace elements include 5 g / L NaCl, 4 g / L MnCl2·4H2O, 1 g / L ZnSO4·7H2O, 0.5 g / L sodium molybdate dihydrate, 4.75 g / L FeCl3·6H2O, 0.575 g / L boric acid, 0.4 g / L CuSO4·5H2O, and 2.038 ml / L concentrated sulfuric acid.

[0016] In one implementation, S5 specifically includes: S51. Resuspend the cells in 20 mM Tris-HCl (pH 7.5) buffer at a ratio of 1:5 (M / V), and disrupt them in a homogenizer at a pressure of more than 70 MPa for 20 min. S52, centrifuge the disrupted liquid at 12000×g for 20 min at 4°C, and collect the supernatant; S53. The supernatant was filtered through a 1 mm filter membrane and then applied to a Ni column for Ni column affinity chromatography purification. The solution was equilibrated with 20 mM Tris-HCl (pH 7.5) buffer, washed with 40 mM imidazole, and eluted with 500 mM imidazole, and the eluate was collected.

[0017] In a second aspect, the present invention further provides a tetanus toxin, which is prepared by any one of the above-mentioned methods for preparing tetanus toxin.

[0018] Beneficial effects: The tetanus subtoxin and preparation method provided by the present invention can achieve a bacterial expression level of 120 g / L by optimizing the fermentation medium and fermentation process, thereby reducing the fermentation cost; a two-step purification method is adopted in the purification process, combined with Ni column affinity chromatography, and isoelectric point precipitation / Q anion exchange / SP ion exchange, and the purity is achieved by two-step purification to reach more than 95%, the expression level reaches 2000 mg / L, the protein expression level is high, and the target protein expression level is improved; the tetanus subtoxin prepared by the present invention can enable horses to produce a strong and effective immune response, has a high level of protective antibodies, and has good immunogenicity; the tetanus subtoxin provided by the present invention can be used as a high-efficiency veterinary immunogen product, used to induce animals to produce high-titer protective antibodies, and is particularly suitable for veterinary tetanus vaccines and the production of antitoxin products for tetanus; at the same time, the tetanus subtoxin, as a safer alternative toxoid vaccine, can be used to reduce allergic reactions that may be caused by conventional tetanus toxoid (TT) vaccines, and at the same time has a high immune efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A flow chart of the steps of the method for preparing tetanus subtoxin provided by the present invention; Figure 2 This is an SDS-PAGE gel image of the purified Tetanus toxin TeNT-Hc (C869A) antigen provided in Example 1; Figure 3 This is the SEC-HPLC spectrum of the purified Tetanus toxin TeNT-Hc (C869A) antigen provided in Example 1.

[0020] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the descriptions of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0022] Specific reference Figure 1 , Figure 1 The present invention provides a method for preparing tetanus toxin, which comprises the following specific steps: S1. Provide or construct a tetanus toxin Hc fragment mutant TeNT-Hc strain; S2. Preparation of primary seeds: Sterilize the seed culture medium in a shake flask, cool to room temperature, inoculate the TeNT-Hc strain into the seed culture medium at a rate of 0.1% to 0.5%, and culture in a shaker at 37°C for 3 to 8 hours until the OD 600 When the value reaches 0.8-1.5, the first-level seed culture solution is obtained; S3. Preparation of secondary seeds: further amplifying the number of strains based on the primary seeds to obtain secondary seed culture fluid; S4, fermentation: the secondary seed culture solution is inoculated into the fermentation tank at a rate of 1% to 5%, and a feed medium is added for feeding. When the bacterial density of the bacteria reaches 30 to 40, IPTG is added to induce expression. When the OD value reaches 70 or above, the fermentation tank is removed from the fermentation tank, the fermentation solution is collected and centrifuged to collect the bacteria; S5, first step of purification: resuspending the bacteria, breaking and centrifuging, collecting the supernatant, passing the supernatant through Ni column affinity chromatography, washing and eluting the impurities, and collecting the eluate; S6. Second step purification: the eluate is subjected to second step purification by isoelectric precipitation or Q anion exchange or SP ion exchange, and the supernatant or eluate is collected to obtain the tetanus subtoxin.

[0023] Specifically, the TeNT-Hc strain is an engineered bacterium containing the tetanus toxin receptor binding region Hc nucleotide sequence. Alternatively, the TeNT-Hc strain can be prepared by providing a C869A mutant sequence, inserting six His-Tags into the sequence, enzymatically digesting it, ligating it to a pET28a vector, and transforming it into BL21(DE3) E. coli.

[0024] Specifically, the seed culture medium includes 5g / L yeast powder, 10g / L peptone and 10g / L NaCl. In S3, the further expansion of the strain quantity specifically includes: placing the seed culture medium into a shake flask for sterilization, cooling it to room temperature, inoculating the first-level seed culture solution into the seed culture medium at a 0.1% to 0.5% inoculum, placing it in a shaker at 37°C for 3 to 8 hours, and culturing it until the OD 600 When the value reaches 0.8~1.5, the secondary seed culture solution is obtained.

[0025] The preparation process for secondary seeds is identical to that for primary seeds, primarily because they are intended to further expand the strain population based on primary seeds. Therefore, their culture medium, inoculum size, and culture conditions are essentially identical, ensuring that the strains maintain the same growth state and metabolic characteristics during the expansion process. By amplifying the strains in stages (primary and secondary seeds), consistency and controllability are ensured during the expansion process. The preparation of secondary seeds, based on primary seeds, further expands the strain population, providing sufficient bacterial load and optimal growth conditions for subsequent fermentation, thereby improving fermentation efficiency and product yield. Furthermore, the phased expansion of strains allows for quality control and testing at each stage, promptly identifying and eliminating contamination issues.

[0026] Specifically, in S4, it specifically includes: S41. Provide a fermentation tank, adjust the volume according to the fermentation medium formula, and sterilize at 110-130°C for 15-25 minutes; S42, the secondary seeds are inoculated into the fermentation tank at an inoculum rate of 1% to 5%, the temperature is controlled at 37°C, the pH is 7.0, and the dissolved oxygen is ≥30%. After the dissolved oxygen rapidly rises to above 60%, feed medium is added for feeding; wherein, the initial feeding rate is 15 g / (L*h), and the dissolved oxygen is controlled to be ≥30% after feeding; S43. When the bacterial density reaches 30-40, start cooling and set the temperature to 16-25°C. S44, add 0.1mM~1mM IPTG for induction, and induce for 8~12h; S45, when the OD value reaches above 70, the tank is unloaded and the fermentation liquid is collected; S46. Centrifuge the fermentation broth and collect the centrifuged bacteria.

[0027] Furthermore, in S41, the fermentation medium composition is: 5 g / L yeast extract, 5 g / L ammonium sulfate, 10 g / L glucose, 3.8 g / L dipotassium hydrogen phosphate, 3.5 g / L anhydrous potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 1 g / L trace elements, 0.075 g / L calcium chloride, and 0.0022 g / L VB1. The feed medium includes 500 g / L glucose, 20 g / L magnesium sulfate, and 1 g / L trace elements. The trace elements include 5g / L NaCl, 4g / L MnCl2·4H2O, 1g / L ZnSO4·7H2O, 0.5g / L sodium molybdate dihydrate, 4.75g / L FeCl3·6H2O, 0.575g / L boric acid, 0.4g / L CuSO4·5H2O, and 2.038ml / L concentrated sulfuric acid. By controlling the temperature, pH, dissolved oxygen and feeding rate during the fermentation process, and performing IPTG induction at an appropriate bacterial density, the optimal growth environment of the strain during the fermentation process is ensured, and the expression of the target protein is increased. The staged seed culture and strict fermentation parameter control ensure the repeatability and stability of the entire preparation process, making this method have high application value in industrial production.

[0028] Furthermore, in S5, it specifically includes: S51. Resuspend the cells in 20 mM Tris-HCl (pH 7.5) buffer at a ratio of 1:5 (M / V), and disrupt them in a homogenizer at a pressure of more than 70 MPa for 20 min. S52, centrifuge the disrupted liquid at 12000×g for 20 min at 4°C, and collect the supernatant; S53. The supernatant was filtered through a 1 mm filter membrane and then applied to a Ni column for Ni column affinity chromatography purification. The solution was equilibrated with 20 mM Tris-HCl (pH 7.5) buffer, washed with 40 mM imidazole, and eluted with 500 mM imidazole, and the eluate was collected.

[0029] Specifically, in S6, the target protein with a purity of more than 95% can be obtained by adjusting the pH to 5.0, performing isoelectric precipitation, and then collecting the supernatant by centrifugation; Alternatively, after purification on a Ni column, apply it to a Q anion exchange column, equilibrate it with 20mM Tris-HCl (pH 8.0), and elute it with a gradient of 0-0.5M NaCl in 20mM Tris-HCl (pH 8.0) buffer. The eluate is the target protein with a purity of more than 95% at a concentration of about 150mM NaCl. Alternatively, after Ni column purification, apply it to an SP cation exchange column, equilibrate with 20mM sodium acetate (pH 4.5) buffer, and elute with a gradient of 0-0.5M NaCl in 20mM sodium acetate (pH 4.5) buffer. Elution at a concentration of approximately 450mM NaCl will result in the eluate containing the target protein with a purity of more than 95%.

[0030] Through two-step purification (Ni column affinity chromatography and isoelectric precipitation or ion exchange chromatography), impurities are effectively removed to obtain high-purity tetanus toxin.

[0031] Among them, isoelectric precipitation is suitable for when the isoelectric point of the target protein is close to neutral (pH 5.0 ~ 6.5). By adjusting the solution pH to the isoelectric point of the target protein, impurities with significantly different charge characteristics can be effectively removed. It is particularly suitable for processing samples with high protein impurities in fermentation broth; Q anion exchange is suitable for when the isoelectric point of the target protein is low (pH <5.0). By using anion exchange medium to capture the protein under alkaline buffer conditions (such as pH 8.0), efficient impurity removal is achieved. It is particularly suitable for cases where the impurity proteins in the sample have a strong negative charge; SP cation exchange is suitable for when the isoelectric point of the target protein is high (pH >6.5). By using cation exchange medium to capture the target protein under acidic buffer conditions (such as pH 4.5), the purity is further improved. It is suitable for removing positively charged impurity proteins in the sample.

[0032] Example 1 S1. Construction of strains: providing an engineered bacterium containing the tetanus toxin receptor binding region Hc nucleotide sequence; S2. Preparation of primary seeds: Sterilize the seed culture medium in a shake flask, cool to room temperature, inoculate the strain into the seed culture medium at a 0.1% inoculum in a clean bench, and culture in a shaker at 37°C for 6 h until the OD 600 When the value reaches 1.3, the first-level seed culture solution is obtained; S3. Preparation of secondary seeds: Sterilize the seed culture medium in a shake flask, cool it to room temperature, inoculate the strain into the seed culture medium at a 0.1% inoculum in a clean bench, and culture it in a shaker at 37°C for 5 h until the OD 600 When the value reaches 1.2, the secondary seed culture solution is obtained; S4. Parameters during the fermentation process: In a 5L fermenter, the volume was adjusted to 3L according to the fermentation medium formula, sterilized at 121℃ for 20min, and the secondary seeds were inoculated into the fermenter at a 3% inoculation rate. The temperature was controlled at 37℃, pH 7.0, and dissolved oxygen ≥ 30%. After the dissolved oxygen quickly rose to above 60%, the initial feeding rate was 15g / (L*h). After feeding, the dissolved oxygen was controlled to ≥ 30% (the feeding rate was adjusted according to the dissolved oxygen feedback). When the bacterial density reached 30, the temperature was lowered and the temperature was set to 16℃. 0.2mM IPTG was added for induction. After 10h of induction, the OD value reached 75, and the tank was unloaded, and 3.3L of fermentation broth was collected. The fermentation broth was centrifuged at a centrifugal force of 12000xg at 4℃ for 20min to collect the bacteria. The wet weight of the strain was 121g / L; S5. Resuspend the cells in 20mM Tris-HCl (pH 7.5) buffer (containing 500mM NaCl and 0.1% Triton X-100) at a 1:5 (M / V) ratio and disrupt in a homogenizer at a pressure of at least 70 MPa for 20 minutes. Centrifuge the disrupted liquid at 12,000xg for 20 minutes at 4°C, and collect the supernatant. Pass the supernatant through a 1mm filter and purify on a Ni column, equilibrated with 20mM Tris-HCl (pH 7.5) buffer, washed with 40mM imidazole, and eluted with 500mM imidazole. Collect the eluate. S6. Adjust the pH to 5.0 and isoelectric precipitation, then centrifuge and collect the supernatant, which is the target protein with a purity of more than 95%; Alternatively, after purification on a Ni column, apply it to a Q anion exchange column, equilibrate it with 20mM Tris-HCl (pH 8.0), and elute it with a gradient of 0-0.5M NaCl in 20mM Tris-HCl (pH 8.0) buffer. The eluate is the target protein with a purity of more than 95% at a concentration of about 150mM NaCl. Alternatively, after Ni column purification, apply it to an SP cation exchange column, equilibrate with 20mM sodium acetate (pH 4.5) buffer, and elute with a gradient of 0-0.5M NaCl in 20mM sodium acetate (pH 4.5) buffer. Elution at a concentration of approximately 450mM NaCl will result in the eluate containing the target protein with a purity of more than 95%.

[0033] Table 1. Data comparison table of Example 1 and comparative example Among them, SEC-HPLC (size exclusion high performance liquid chromatography) is an HPLC technology based on molecular size separation. The comparative example is the fermentation method and data of Example 1 in Patent 201611256457.

[0034] Table 2. Different antigen purification processes of Examples 1 to 3 Combine Figure 2~Figure 3 As well as the data results in Table 1 and Table 2, Figure 2 This is an SDS-PAGE gel image of the purified Tetanus toxin TeNT-Hc (C869A) antigen provided in Example 1; Figure 3 The SEC-HPLC spectrum of the purified TeNT-Hc (C869A) antigen of the tetanus subtoxin provided in Example 1 shows that the preparation method of tetanus subtoxin provided by the present invention can achieve a bacterial expression level of 120 g / L by optimizing the fermentation medium and fermentation process, thereby reducing the fermentation cost; a two-step purification method is adopted in the purification process, combining Ni column affinity chromatography, and isoelectric point precipitation / Q anion exchange / SP ion exchange, and a purity of more than 95% is achieved through two-step purification, and the expression level reaches 2000 mg / L, with high protein expression.

[0035] Example 4: Horse Immunization Experiment Experimental Materials: Horses: Purchased from herders in Datong, Qinghai Province, healthy stallions without natural tetanus antibodies were selected, aged 4 to 10 years, with an average age of 6.5 years, and weighing 250 to 400 kg, with an average weight of 328.3 kg.

[0036] Incomplete Freund's adjuvant: prepared by mixing liquid paraffin and lanolin in a volume ratio of 2:1.

[0037] Antigens and adjuvants: TeNT-Hc antigen, molecular weight 50kD, is the purified tetanus toxin TeNT-Hc (C869A) antigen prepared by Example 1, which is mixed with incomplete Freund's adjuvant in a 1:1 ratio to form an oil-in-water emulsion with a protein content of 0.625 mg / ml.

[0038] Tetanus toxin standard and anti-tetanus serum national standard were purchased from the China Food and Drug Administration. ICR mice: 17-19 g, half male and half female.

[0039] Experimental methods: 1. Horse grouping, immunization procedures, and serum and plasma separation Six horses without natural antibodies and in good age, weight, and health were selected and divided into two groups of three horses each. At different stages of immunization, horses were vaccinated with different doses of TeNT-Hc and TT via multiple intramuscular injections in the neck and back, for a total of three cycles. The first cycle consisted of two primary immunizations, administered seven days apart. Seven booster immunizations were administered 56 days after the primary immunization, each administered seven days apart. A second cycle of immunizations was administered 16 days after the last booster immunization, for a total of three cycles, each administered seven days apart. A third cycle of immunizations was administered 18 days after the last dose of the second cycle, for a total of three cycles, each administered seven days apart. Specific antigen types and immunization doses are shown in Table 3. Serum and plasma were collected separately at different stages of immunization. Serum was collected naturally via jugular vein blood collection, while plasma was collected using a single plasmapheresis device from the jugular vein.

[0040] Table 3. Horse groups and antigen immunization scheme Antibody level testing: Antibody titers were determined using ELISA. 100 μL / well of TeNT-Hc 1 μg / ml was coated on a 96-well ELISA plate at 4°C overnight. The plates were then washed five times with PBST, each time for 5 minutes.

[0041] The stock solution of the antibody-breaking standard, 9 IU / mL, was diluted 2-fold in 10 gradients: 900 mIU, 450 mIU, 225 mIU, 112.5 mIU, 56.25 mIU, 28.13 mIU, 14.06 mU, 7.03 mIU, 3.5 mIU, 0. Each gradient was diluted in duplicate.

[0042] Add horse serum or plasma, serially diluted from 1:20,000, and incubate at 37°C for 1 hour. Wash five times with PBST, 5 minutes each. Add HRP-anti-horse secondary antibody and react at 37°C for 30 minutes. Wash five times with PBST, 5 minutes each. Add TMB colorimetric solution (50 μL / well). After development, terminate the reaction with 50 μL / well of 2M sulfuric acid. Measure absorbance at 450 nm using a microplate reader. Results are shown in Table 4.

[0043] Table 4. Serum (plasma) titer detection after immunization of horses (ELISA method) According to the data in the table, the tetanus subtoxin provided by the present invention can be combined with or replace the attenuated tetanus toxin toxoid (TT) as a highly effective antigen, so that horses can produce a strong and effective immune response with a high level of protective antibodies, and can provide a tetanus vaccine product that is non-toxic, has low allergic reactions, is reasonably priced, and is of high quality.

[0044] The present invention focuses on the construction and purification of the TeNT-Hc fragment, which significantly reduces toxicity while still maintaining its neutralizing immunogenicity, as described below: Comparison of tetanus toxin (TeNT) toxicity 50 Analysis of the acute toxicity test mechanism In summary, the tetanus subtoxin and its preparation method provided by the present invention can achieve a bacterial expression level of 120 g / L by optimizing the fermentation medium and fermentation process, thereby reducing the fermentation cost; a two-step purification method is adopted in the purification process, combined with Ni column affinity chromatography, and isoelectric point precipitation / Q anion exchange / SP ion exchange, and a purity of more than 95% is achieved through two-step purification, and the expression level reaches 2000 mg / L, with high protein expression; the tetanus subtoxin prepared by the present invention enables horses to produce a strong and effective immune response, has a high level of protective antibodies, good immunogenicity, and is almost non-toxic, and will not cause liver disease and death in horses. The tetanus subtoxin prepared by the present invention can enable horses to produce a strong and effective immune response, has a high level of protective antibodies, and has good immunogenicity; in addition, the tetanus subtoxin can be used as a high-efficiency veterinary immunogen product to induce animals to produce high-titer protective antibodies, and is particularly suitable for veterinary tetanus vaccines and the production of antitoxin products for tetanus; at the same time, the tetanus subtoxin, as a safer alternative toxoid vaccine, can be used to reduce allergic reactions that may be caused by conventional tetanus toxoid (TT) vaccines, and at the same time has a higher immune efficacy.

[0045] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing tetanus toxin, characterized in that: The specific steps include: S1. Provide or construct a tetanus toxin Hc fragment mutant TeNT-Hc strain; S2. Preparation of primary seeds: Sterilize the seed culture medium in a shake flask, cool to room temperature, inoculate the TeNT-Hc strain into the seed culture medium at a rate of 0.1% to 0.5%, and culture in a shaker at 37°C for 3 to 8 hours until OD 600 When the value reaches 0.8-1.5, the first-grade seed culture solution is obtained; S3, preparing secondary seeds: further amplifying the number of strains on the basis of the primary seeds to obtain secondary seed culture solution; S4, fermentation: the secondary seed culture solution is inoculated into a fermentation tank at an inoculation rate of 1% to 5%, and a feed medium is added for feeding. When the bacterial density of the bacteria reaches 30 to 40, IPTG is added to induce expression. When the OD value reaches 70 or more, the fermentation solution is collected and centrifuged to collect the bacteria; S5, first step of purification: resuspending, crushing and centrifuging the bacteria, collecting the supernatant, passing the supernatant through Ni column affinity chromatography, washing and eluting, and collecting the eluate; S6. Second step purification: the eluate is subjected to second step purification by isoelectric precipitation or Q anion exchange or SP ion exchange, and the supernatant or eluate is collected to obtain the tetanus subtoxin.

2. The method for preparing tetanus toxin according to claim 1, characterized in that: In S1, the TeNT-Hc strain is an engineered bacterium containing the tetanus toxin receptor binding region Hc nucleotide sequence.

3. The method for preparing tetanus toxin according to claim 1, characterized in that: In S2, the seed culture medium includes 5 g / L yeast powder, 10 g / L peptone and 10 g / L NaCl.

4. The method for preparing tetanus toxin according to claim 1, characterized in that: In S3, the further expansion of the strain quantity specifically includes: placing the seed culture medium in a shake flask for sterilization, cooling to room temperature, inoculating the primary seed culture solution into the seed culture medium at a 0.1% to 0.5% inoculation rate, and placing the culture medium in a shaker at 37°C for 3 to 8 hours until the OD 600 When the value reaches 0.8~1.5, the secondary seed culture solution is obtained.

5. The method for preparing tetanus toxin according to claim 1, characterized in that: In S4, it specifically includes: S41, provide a fermentation tank, adjust the volume according to the fermentation medium formula, and sterilize at 110-130°C for 15-25 minutes; S42, the secondary seeds are inoculated into the fermentation tank at a rate of 1% to 5%, the temperature is controlled at 37°C, the pH is 7.0, and the dissolved oxygen is ≥30%. When the dissolved oxygen rises rapidly to above 60%, feed medium is added for feeding; wherein, the initial feeding rate is 15 g / (L*h), and the dissolved oxygen is controlled to be ≥30% after feeding; S43, when the bacterial density reaches 30-40, start cooling, and the temperature is set to 16℃~25℃; S44, add 0.1mM~1mM IPTG for induction, induction for 8~12h; S45, when the OD value reaches above 70, the tank is lowered to collect the fermentation liquid; S46, centrifuging the fermentation broth and collecting the bacterial cells after centrifugation.

6. The method for preparing tetanus toxin according to claim 1, characterized in that: In S4 and S41, the fermentation medium composition was: yeast extract 5 g / L, ammonium sulfate 5 g / L, glucose 10 g / L, dipotassium hydrogen phosphate 3.8 g / L, anhydrous potassium dihydrogen phosphate 3.5 g / L, magnesium sulfate heptahydrate 1 g / L, trace elements 1 g / L, calcium chloride 0.075 g / L, and VB1 0.0022 g / L.

7. The method for preparing tetanus toxin according to claim 1, characterized in that: In S4, the feed medium includes 500 g / L glucose, 20 g / L magnesium sulfate, and 1 g / L trace elements.

8. The method for preparing tetanus toxin according to claim 6 or 7, characterized in that: In S4, the trace elements include 5g / L NaCl, 4g / L MnCl2·4H2O, 1g / L ZnSO4·7H2O, 0.5g / L sodium molybdate dihydrate, 4.75g / L FeCl3·6H2O, 0.575g / L boric acid, 0.4g / L CuSO4·5H2O, and 2.038ml / L concentrated sulfuric acid.

9. The method for preparing tetanus toxin according to claim 1, characterized in that: In S4 and S5, specifically including: S51, resuspend the bacteria in 20mM Tris-HCl (pH7.5) buffer at a ratio of 1:5 (M / V), break them in a homogenizer at a pressure of more than 70Mpa, and circulate for 20min; S52, centrifuge the broken liquid at 12000xg for 20 min at 4°C and collect the supernatant; S53. The supernatant is filtered through a 1 mm filter membrane and then placed on a Ni column for Ni column affinity chromatography purification. The supernatant is balanced with 20 mM Tris-HCl (pH 7.5) buffer, washed with 40 mM imidazole, eluted with 500 mM imidazole, and the eluate is collected.

10. A tetanus toxin prepared by the method according to any one of claims 1 to 9.

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