CN119350461A - Tetanus subtoxin and preparation method thereof - Google Patents

Abstract

The present invention relates to a tetanus toxin and a preparation method thereof. The preparation method specifically includes: 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 of 0.1% to 0.5%, shaking and culturing until the OD ₆₀₀ value reaches 0.8 to 1.5, and obtaining a primary seed culture solution; further expanding the number of strains on the basis of the primary seed to obtain a secondary seed culture solution; fermenting; after the bacterial cell is resuspended, crushed and centrifuged, the supernatant is collected, the supernatant is passed through a Ni column affinity chromatography, and the eluate is collected after washing and eluting; the eluate is purified in a second step by isoelectric precipitation or Q anion exchange or SP ion exchange, and the supernatant or eluate is collected to obtain the tetanus toxin. The present invention achieves a purity of more than 95% by two-step purification, while increasing the target protein expression amount.

Description

Tetanus toxoid and preparation method thereof

Technical Field

The invention relates to the technical field of biological products, in particular to tetanus toxoid and a preparation method thereof.

Background

Tetanus is a serious disease caused by clostridium tetani infection. Clostridium tetani breeds under hypoxic conditions and produces tetanus neurotoxin. The toxins enter the nervous system through the blood, act on the central nervous system, inhibit neurotransmitter release, and cause interruption of the transmission of information between nerves and muscles, thereby causing serious respiratory dysfunction and hemodynamic dysfunction. Tetanus toxin is extremely deadly, and can die about 100 nanograms. About 100 thousands of tetanus cases are worldwide each year, with mortality rates up to 50% and neonatal tetanus in developing countries even up to 90%. Therefore, development of efficient and safe tetanus preventive and therapeutic drugs is an important point of scientific research in various countries.

Tetanus toxin is cleaved by proteases into disulfide-linked light and heavy chains, whose functions can be divided into A, B, C parts (each having a molecular weight of 50 kD) which act as binding, infusion and paralysis, respectively, thereby inhibiting neurotransmitter release. The natural C fragment (Hc) is the C end of the heavy chain, retains various properties of combining the complete toxin and ganglioside, has the immune titer equivalent to that of the toxin, has lower allergenicity, and can be used for preparing subunit vaccines.

The expression of tetanus Hc section is improved in many researches at home and abroad, and a soluble expression product of recombinant Hc protein is obtained by utilizing escherichia coli, for example, a recombinant bacterium for expressing tetanus toxin subunit vaccine Hc in vitro and a recombinant expression method are disclosed in patent application No. 200910135972, but the expression level is low, the expression quantity of soluble target protein is only 300mg/L, and a method for improving the expression quantity by high-density fermentation is disclosed in patent application No. 201611256457, and the expression quantity of the soluble target protein can reach 800mg/L. However, the purification process is complicated, three column chromatography is used, wherein the supernatant is subjected to anion exchange by a QFF column, hydrophobic chromatography by a phenyl hydrophobic column, and cation exchange by an SP column.

The existing research has successfully expressed recombinant Hc protein by using escherichia coli, but has the problems of low expression level, complex purification process and the like.

Accordingly, improvements are needed in the art.

Disclosure of Invention

In the prior art, the recombinant Hc protein has been successfully expressed by using escherichia coli, but the problems of low expression level or complex purification process exist, so the invention provides tetanus toxoid and a preparation method thereof for solving the problems.

To achieve the above object, in a first aspect, the present invention provides a process for the preparation of tetanus toxoid, comprising the following specific steps:

S1, providing or constructing a tetanus toxin Hc fragment mutant TeNT-Hc strain;

S2, preparing first-stage seeds, namely placing a seed culture medium into a shake flask for sterilization, cooling to room temperature, inoculating the TeNT-Hc strain into the seed culture medium according to the inoculation amount of 0.1% -0.5%, placing the TeNT-Hc strain into a shaking table, and carrying out constant-temperature shaking culture for 3-8 hours at 37 ℃ until the OD ₆₀₀ value reaches 0.8-1.5, so as to obtain first-stage seed culture solution;

s3, preparing secondary seeds, namely further amplifying the number of strains on the basis of the primary seeds to obtain secondary seed culture solution;

S4, fermenting, namely inoculating the secondary seed culture solution into a fermentation tank according to an inoculum size of 1% -5%, adding a feed medium for feeding, adding IPTG for induction expression when the bacterial density of the bacterial reaches 30% -40, performing tank unloading operation when the OD value reaches above 70, collecting fermentation liquor, centrifuging, and collecting the bacterial;

S5, purifying in the first step, namely re-suspending, crushing and centrifuging the bacteria, collecting supernatant, subjecting the supernatant to Ni column affinity chromatography, eluting, and collecting eluent;

S6, purifying the eluent in the second step through isoelectric point precipitation or Q anion exchange or SP ion exchange, and collecting supernatant or eluent to obtain the tetanus toxoid.

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

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

In an implementation mode, in S3, the further amplification of the bacterial strain number specifically comprises the steps of filling a seed culture medium into a shake flask for sterilization, cooling to room temperature, inoculating the primary seed culture solution into the seed culture medium according to the inoculation amount of 0.1% -0.5%, and placing the primary seed culture solution into a shaking table for shaking culture at a constant temperature of 37 ℃ for 3-8 hours until the OD ₆₀₀ value reaches 0.8-1.5, so as to obtain the secondary seed culture solution.

In one implementation, in S4, specifically includes:

s41, providing a fermentation tank, carrying out constant volume according to a fermentation medium formula, and sterilizing for 15-25 min at 110-130 ℃;

s42, inoculating the secondary seeds into a fermentation tank according to an inoculum size of 1% -5%, controlling the temperature to be 37℃, pH and 7.0, controlling the dissolved oxygen to be more than or equal to 30%, and adding a feed medium to feed when the dissolved oxygen rises to more than 60% rapidly, wherein the initial feed speed is 15 g/(L.times.h), and controlling the dissolved oxygen to be more than or equal to 30% after feeding;

s43, when the bacterial density reaches 30-40, starting to cool, wherein the temperature is set to be 16-25 ℃;

S44, adding 0.1 mM-1 mM MIPTG for induction for 8-12 h;

s45, when the OD value reaches more than 70, carrying out tank discharging operation, and collecting fermentation liquor;

S46, centrifuging the fermentation liquor, and collecting the centrifuged thalli.

In one implementation, in S41, the fermentation medium consists of 5g/L yeast extract, 5g/L ammonium sulfate, 10g/L glucose, 3.8g/L dipotassium hydrogen phosphate, 3.5g/L anhydrous potassium dihydrogen phosphate, 1g/L magnesium sulfate heptahydrate, 1g/L trace elements, 0.075g/L calcium chloride, and 10.0022g/L VB10.

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

In one implementation, the trace elements include 5g/L NaCl, 4g/L MnCl ₂·4H₂ O, 1g/L ZnSO ₄·7H₂ O,0.5g/L sodium molybdate dihydrate, 4.75g/L FeCl ₃·6H₂ O, 0.575g/L boric acid, 0.4g/L CuSO4.5H2 ₂ O, and 2.038ml/L concentrated sulfuric acid.

In one implementation, in S5, specifically includes:

s51, re-suspending the thalli according to 1:5 (M/V) and 20mM Tris-HCl (pH 7.5) buffer solution, crushing the thalli in a homogenizer at a pressure of more than 70MPa, and circulating the thalli for 20min;

S52, centrifuging the crushed liquid at a centrifugal force of 12000xg and a temperature of 4 ℃ for 20min to collect supernatant;

S53, purifying the supernatant by Ni column affinity chromatography on a Ni column after passing through a 1mM filter membrane, balancing with 20mM Tris-HCl (pH 7.5) buffer solution, washing impurities with 40mM imidazole, eluting with 500mM imidazole, and collecting eluent.

In a second aspect, the invention also provides a tetanus toxoid obtainable by a process for the preparation of a tetanus toxoid according to any one of the preceding claims.

The tetanus toxoid and the preparation method thereof have the beneficial effects that the bacterial body expression amount can reach 120g/L through optimizing a fermentation culture medium and a fermentation process, the fermentation cost is reduced, a two-step purification method is adopted in the purification process, the Ni column affinity chromatography and isoelectric precipitation/Q anion exchange/SP ion exchange are combined, the purity is over 95% through two-step purification, the expression amount reaches 2000mg/L, the protein expression amount is high, the target protein expression amount is improved, the tetanus toxoid prepared by the method can enable horses to generate strong and effective immune response, has higher protective antibody level and good immunogenicity, the tetanus toxoid provided by the invention can be used as a veterinary high-efficiency immunogen product for inducing animals to generate high-titer protective antibodies, is particularly suitable for tetanus vaccines and tetanus antitoxin products aiming at tetanus vaccines, and simultaneously, the tetanus toxoid can be used as a substitute toxoid vaccine with higher safety and can be used for reducing possible induction of conventional Tetanus Toxoid (TT) and has higher immune effect.

Drawings

FIG. 1 is a flow chart of steps of a process for preparing tetanus toxoid provided by the invention;

FIG. 2 is a SDS-PAGE gel of the TeNT-Hc (C869A) antigen after purification of tetanus toxoid provided in example 1;

FIG. 3 is a SEC-HPLC profile of the TeNT-Hc (C869A) antigen after purification of tetanus toxoid provided in example 1.

The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. Furthermore, the descriptions of the terms "one embodiment," "some embodiments," "examples," "particular examples," or "some examples," etc., described below mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily for the same embodiment or example. The technical features of the respective embodiments of the present invention may be combined with each other as long as they do not collide with each other.

Referring specifically to fig. 1, fig. 1 is a flow chart of steps of a preparation method of tetanus toxoid provided by the invention. The invention provides a preparation method of tetanus toxoid, which comprises the following specific steps:

S1, providing or constructing a tetanus toxin Hc fragment mutant TeNT-Hc strain;

S2, preparing first-stage seeds, namely placing a seed culture medium into a shake flask for sterilization, cooling to room temperature, inoculating the TeNT-Hc strain into the seed culture medium according to the inoculation amount of 0.1% -0.5%, placing the TeNT-Hc strain into a shaking table, and carrying out constant-temperature shaking culture for 3-8 hours at 37 ℃ until the OD ₆₀₀ value reaches 0.8-1.5, so as to obtain first-stage seed culture solution;

s3, preparing secondary seeds, namely further amplifying the number of strains on the basis of the primary seeds to obtain secondary seed culture solution;

S4, fermenting, namely inoculating the secondary seed culture solution into a fermentation tank according to an inoculum size of 1% -5%, adding a feed medium for feeding, adding IPTG for induction expression when the bacterial density of the bacterial reaches 30% -40, performing tank unloading operation when the OD value reaches above 70, collecting fermentation liquor, centrifuging, and collecting the bacterial;

S5, purifying in the first step, namely re-suspending, crushing and centrifuging the bacteria, collecting supernatant, subjecting the supernatant to Ni column affinity chromatography, eluting, and collecting eluent;

S6, purifying the eluent in the second step through isoelectric point precipitation or Q anion exchange or SP ion exchange, and collecting supernatant or eluent to obtain the tetanus toxoid.

Specifically, the TeNT-Hc strain is an engineering bacterium containing a tetanus toxin receptor binding region Hc nucleotide sequence. Or the TeNT-Hc strain can be transferred into BL21 (DE 3) escherichia coli by providing a C869A mutant sequence, inserting 6 His-tags into the sequence, connecting the sequence to a PET28a carrier after enzyme digestion.

Specifically, the seed culture medium comprises 5g/L yeast powder, 10g/L peptone and 10g/L NaCl. In S3, the number of the further amplified strains specifically comprises the steps of filling a seed culture medium into a shake flask for sterilization, cooling to room temperature, inoculating the primary seed culture solution into the seed culture medium according to the inoculation amount of 0.1% -0.5%, placing the seed culture medium into a shaking table, and carrying out constant-temperature shaking culture for 3-8 h at 37 ℃ until the OD ₆₀₀ value reaches 0.8-1.5, so as to obtain the secondary seed culture solution.

The preparation process of the secondary seeds is the same as that of the primary seeds, mainly because the purpose of the secondary seeds is to further amplify the number of the strains on the basis of the primary seeds, and therefore, the culture medium, the inoculation amount and the culture conditions of the secondary seeds are basically consistent, so that the strains can keep the same growth state and metabolic characteristics in the amplification process. By amplifying the strain (primary seed and secondary seed) in stages, uniformity and controllability of the strain during the amplification process are ensured. The preparation of the secondary seeds is based on the primary seeds, the number of strains is further amplified, and enough bacterial quantity and good growth state are provided for subsequent fermentation, so that the fermentation efficiency and the product yield are improved. In addition, the strain can be amplified in stages to perform quality control and detection at each stage, so that pollution problems can be timely found and eliminated.

Specifically, in S4, specifically includes:

s41, providing a fermentation tank, carrying out constant volume according to a fermentation medium formula, and sterilizing for 15-25 min at 110-130 ℃;

s42, inoculating the secondary seeds into a fermentation tank according to an inoculum size of 1% -5%, controlling the temperature to be 37℃, pH and 7.0, controlling the dissolved oxygen to be more than or equal to 30%, and adding a feed medium to feed when the dissolved oxygen rises to more than 60% rapidly, wherein the initial feed speed is 15 g/(L.times.h), and controlling the dissolved oxygen to be more than or equal to 30% after feeding;

s43, when the bacterial density reaches 30-40, starting to cool, wherein the temperature is set to be 16-25 ℃;

S44, adding 0.1 mM-1 mM MIPTG for induction for 8-12 h;

s45, when the OD value reaches more than 70, carrying out tank discharging operation, and collecting fermentation liquor;

S46, centrifuging the fermentation liquor, and collecting the centrifuged thalli.

Further, in S41, the fermentation medium consists of 5g/L of yeast extract powder, 5g/L of ammonium sulfate, 10g/L of glucose, 3.8g/L of dipotassium hydrogen phosphate, 3.5g/L of anhydrous potassium dihydrogen phosphate, 1g/L of magnesium sulfate heptahydrate, 1g/L of trace elements, 0.075g/L of calcium chloride and 10.0022g/L of VB10. The feed medium comprises 500g/L glucose, 20g/L magnesium sulfate and 1g/L trace elements. The microelements comprise 5g/L NaCl, 4g/L MnCl ₂·4H₂ O, 1g/L ZnSO ₄·7H₂ O,0.5g/L sodium molybdate dihydrate, 4.75g/L FeCl ₃·6H₂ O, 0.575g/L boric acid, 0.4g/L CuSO4.5H2 ₂ O and 2.038ml/L concentrated sulfuric acid. By controlling the temperature, pH, dissolved oxygen and feeding speed in the fermentation process and performing IPTG induction under proper bacterial density, the optimal growth environment of the strain in the fermentation process is ensured, and the expression level of target protein is improved. The repeatability and stability of the whole preparation process are ensured by the staged seed culture and strict fermentation parameter control, so that the method has higher application value in industrial production.

Further, in S5, specifically includes:

s51, re-suspending the thalli according to 1:5 (M/V) and 20mM Tris-HCl (pH 7.5) buffer solution, crushing the thalli in a homogenizer at a pressure of more than 70MPa, and circulating the thalli for 20min;

S52, centrifuging the crushed liquid at a centrifugal force of 12000xg and a temperature of 4 ℃ for 20min to collect supernatant;

S53, purifying the supernatant by Ni column affinity chromatography on a Ni column after passing through a 1mM filter membrane, balancing with 20mM Tris-HCl (pH 7.5) buffer solution, washing impurities with 40mM imidazole, eluting with 500mM imidazole, and collecting eluent.

Specifically, in S6, the supernatant is collected by centrifugation after pH is adjusted to 5.0 isoelectric point precipitation, namely the target protein with the purity of more than 95%;

or purifying with Ni column, loading onto Q anion exchange column, balancing with 20mM Tris-HCl (pH 8.0), gradient eluting with 20mM Tris-HCl (pH 8.0) buffer solution of 0-0.5M NaCl, eluting with about 150mM NaCl concentration, and collecting eluate as target protein with purity of more than 95%;

or purifying with Ni column, balancing with 20mM sodium acetate (pH 4.5) buffer, and gradient eluting with 0-0.5M NaCl 20mM sodium acetate (pH 4.5) buffer to obtain target protein with purity of above 95% at about 450mM NaCl concentration.

Through two-step purification (Ni column affinity chromatography and isoelectric precipitation or ion exchange chromatography), impurities are effectively removed, and the tetanus toxoid with high purity is obtained.

The isoelectric point precipitation is suitable for removing impurities with obvious difference in charge characteristics by adjusting the pH value of a solution to the isoelectric point of target protein when the isoelectric point of the target protein is close to neutral (pH 5.0-6.5), is particularly suitable for treating samples with higher protein impurities in fermentation liquor, the Q anion exchange is suitable for removing impurities with high efficiency by capturing the proteins by utilizing an anion exchange medium under the condition of alkaline buffer (such as pH 8.0) when the isoelectric point of the target protein is lower (pH < 5.0), and is particularly suitable for removing positively charged impurity proteins in samples when the isoelectric point of the target protein is higher (pH > 6.5), and the SP cation exchange is suitable for capturing the target protein by utilizing a cation exchange medium under the condition of acidic buffer (such as pH 4.5), so that the purity is further improved and the positively charged impurity proteins in the samples are suitable for removing.

Example 1

S1, constructing a strain, namely providing engineering bacteria containing a nucleotide sequence of a tetanus toxin receptor binding region Hc;

S2, preparing first-stage seeds, namely placing a seed culture medium into a shake flask for sterilization, cooling to room temperature, inoculating a strain into the seed culture medium according to 0.1% of inoculation amount in an ultra-clean workbench, placing the strain into a shaking table for constant-temperature shaking culture at 37 ℃ for 6 hours until the OD ₆₀₀ value reaches 1.3, and obtaining first-stage seed culture solution;

S3, preparing secondary seeds, namely placing a seed culture medium into a shake flask for sterilization, cooling to room temperature, inoculating a strain into the seed culture medium according to 0.1% of inoculation amount in an ultra-clean workbench, placing the strain into a shaking table for constant-temperature shaking culture at 37 ℃ for 5 hours until the OD ₆₀₀ value reaches 1.2, and obtaining secondary seed culture solution;

S4, parameters in the fermentation process are that in a 5L fermentation tank, the volume is fixed to 3L according to a fermentation medium formula, sterilization is carried out for 20min at 121 ℃, the secondary seeds are inoculated into the fermentation tank according to the inoculation quantity of 3 percent, the temperature is controlled to be 37 ℃ and pH7.0, dissolved oxygen is more than or equal to 30 percent, when the dissolved oxygen is rapidly increased to more than 60 percent, the initial feeding speed is 15 g/(L.times.h), and after feeding, the dissolved oxygen is controlled to be more than or equal to 30 percent (the feeding speed is adjusted according to the feedback of the dissolved oxygen). When the bacterial density reached 30, the temperature was reduced to 16℃and induction was carried out by adding 0.2mM IPTG. And (3) inducing for 10 hours until the OD value reaches 75, performing tank discharging operation, and collecting 3.3L of fermentation broth. The fermentation broth was centrifuged at 12000Xg for 20min at a centrifugal force of 4℃to collect the cells. The wet weight of the strain is 121g/L;

S5, re-suspending the bacterial cells according to a ratio of 1:5 (M/V) and 20mM Tris-HCl (pH 7.5) buffer solution (containing 500mM NaCl and 0.1% triton X-100), crushing the bacterial cells in a homogenizer at a pressure of more than 70MPa, and circulating the bacterial cells for 20min. The crushed solution was centrifuged at 12000Xg at 4℃for 20min to collect the supernatant. Purifying the supernatant with a 1mM filter membrane, performing Ni column chromatography, balancing with 20mM Tris-HCl (pH 7.5) buffer solution, washing impurities with 40mM imidazole, eluting with 500mM imidazole, and collecting eluate;

S6, regulating the pH value to be 5.0, performing isoelectric precipitation, and centrifuging to collect supernatant, namely the target protein with the purity of more than 95%;

Or purifying with Ni column, loading onto Q anion exchange column, balancing with 20mM Tris-HCl (pH 8.0), gradient eluting with 20mM Tris-HCl (pH 8.0) buffer solution of 0-0.5M NaCl, eluting with about 150mM NaCl concentration, and collecting eluate with purity of above 95%;

Or purifying with Ni column, balancing with 20mM sodium acetate (pH 4.5) buffer, and gradient eluting with 0-0.5M NaCl 20mM sodium acetate (pH 4.5) buffer to obtain target protein with purity of above 95% at about 450mM NaCl concentration.

Table 1, data comparison table of example 1 and comparative example

Among them, SEC-HPLC (size exclusion high performance liquid chromatography) is an HPLC technique based on molecular size separation, and the fermentation method and data of example 1 in patent 201611256457 are presented in comparative example.

Table 2, different antigen purification processes of examples 1-3

According to the data results of the figures 2-3 and the tables 1 and 2, the figure 2 is a SDS-PAGE gel diagram of the TeNT-Hc (C869A) antigen after purification of the tetanus toxoid provided in the example 1, and the figure 3 is a SEC-HPLC (SEC-HPLC) graph of the TeNT-Hc (C869A) antigen after purification of the tetanus toxoid provided in the example 1, the preparation method of the tetanus toxoid provided by the invention can achieve 120g/L of bacterial expression quantity by optimizing a fermentation medium and a fermentation process, the fermentation cost is reduced, and the purification process adopts a two-step purification method, combines Ni column affinity chromatography and isoelectric precipitation/Q anion exchange/SP ion exchange, achieves more than 95% purity by two-step purification, and has the expression quantity of 2000mg/L and high protein expression quantity.

Example 4, horse immunization experiment

Experimental materials:

Horse is purchased from herding households in Qinghai Datong area, healthy male horses without tetanus natural antibodies are selected, the average age is 6.5 years, the weight is 250-400 kg, and the average weight is 328.3kg.

The Freund's incomplete adjuvant is prepared by mixing liquid paraffin and lanolin according to the volume ratio of 2:1.

Antigen and adjuvant:

The TeNT-Hc antigen, having a molecular weight of 50kD, was purified from tetanus toxoid prepared by example 1 and was mixed with incomplete Freund's adjuvant 1:1 to form a water-in-oil emulsion having a protein content of 0.625mg/m 1.

Tetanus standard toxin and anti-tetanus serum national standard are purchased from 17-19g of ICR mice of Chinese food and drug inspection institute, and male and female halves.

The experimental method comprises the following steps:

1. Horse grouping, immunization program, serum-plasma separation

6 Horses without natural antibodies and with good age, weight and health condition are selected and divided into 2 groups of 3 horses. In different stages of immunization, different doses of TeNT-Hc and TT are adopted to perform horse immunization, the immunization mode is neck and back multipoint intramuscular injection, three-pass immunization is performed, the first pass comprises basic immunization for 2 times, 7 days at intervals, 7 times of boosting immunization are performed after the basic immunization for 56 days, 7 days at intervals, the second pass is performed after 16 days after the last boosting immunization, 3 times at intervals, 7 days at intervals, the third pass is performed after the last needle immunization for 18 days at intervals, and 3 times at intervals, and 7 days at intervals. Specific antigen species and immunizing doses are shown in Table 3. The serum and the plasma are separated and collected respectively at different stages of immunization, horse serum is naturally separated by jugular vein blood sampling, and horse plasma is collected by adopting a single-slurry collector jugular vein.

TABLE 3 horse grouping and antigen immunization protocol

Antibody level detection:

Determination of antibody titres was performed by ELISA, teNT-Hc 1ug/ml,100 uL/well coated 96 well ELISA plate, 4℃overnight. Wash 5 times with wash buffer PBST, 5 min/time.

Breaking the stock solution of the anti-standard substance, 9IU/mL, and diluting according to 2 times of dilution and 10 gradients. 900mIU, 450mIU, 225mIU, 112.5mIU, 56.25mIU, 28.13mIU, 14.06mU, 7.03mIU, 3.5mIU, 0, 2 replicate wells per gradient.

Horse serum or plasma diluted in gradient starting at 1:20000 was added and incubated at 37 ℃ for 1h. PBST was washed 5 times, 5 min/time. HRP-anti Ma Er antibody was added and reacted at 37℃for 30min. PBST was washed 5 times, 5 min/time. TMB color development solution (50. Mu.L/well) was added, and after color development, the light absorption was measured at 450nm using a 50. Mu.L/well 2M sulfuric acid stop microplate reader. The detection results are shown in Table 4.

TABLE 4 serum (plasma) titre assay after immunization of horses (ELISA method)

According to the table data, the tetanus toxoid provided by the invention can be matched with or replace toxoid (TT) of the tetanus toxin subjected to attenuation treatment as a high-efficiency antigen, so that a horse generates strong and effective immune response, has higher protective antibody level, and can provide a tetanus vaccine product with no toxicity, low anaphylactic reaction, reasonable price and high quality.

The invention focuses on the construction and purification of the TeNT-Hc fragment, which significantly reduces toxicity but still maintains the neutralization immunogenicity, and is specifically described as follows:

Tetanus toxin (TeNT) toxicity comparison

₅₀ In conclusion, the tetanus toxoid and the preparation method thereof provided by the invention can reach 120g/L by optimizing a fermentation culture medium and a fermentation process, reduce fermentation cost, and achieve more than 95% purity by adopting a two-step purification method in combination with Ni column affinity chromatography and isoelectric precipitation/Q anion exchange/SP ion exchange in the purification process, wherein the expression level reaches 2000mg/L, and the protein expression level is high. The tetanus toxoid prepared by the method can enable horses to generate strong and effective immune response, has higher protective antibody level and good immunogenicity, can be used as a veterinary efficient immunogen product for inducing animals to generate high-titer protective antibodies, is particularly suitable for veterinary tetanus vaccines and antitoxin products aiming at tetanus, and can be used as a substitute toxoid vaccine with higher safety, can be used for reducing anaphylactic reaction possibly caused by conventional Tetanus Toxoid (TT) vaccines, and has higher immune efficacy.

The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes using the descriptions and drawings of the present invention or directly or indirectly applied to other related technical fields are included in the scope of the invention.

Claims (10)

1. The preparation method of tetanus toxoid is characterized by comprising the following specific steps:

S1, providing or constructing a tetanus toxin Hc fragment mutant TeNT-Hc strain;

S2, preparing first-stage seeds, namely placing a seed culture medium into a shake flask for sterilization, cooling to room temperature, inoculating the TeNT-Hc strain into the seed culture medium according to the inoculation amount of 0.1% -0.5%, placing the TeNT-Hc strain into a shaking table, and carrying out constant-temperature shaking culture for 3-8 hours at 37 ℃ until the OD ₆₀₀ value reaches 0.8-1.5, so as to obtain first-stage seed culture solution;

s3, preparing secondary seeds, namely further amplifying the number of strains on the basis of the primary seeds to obtain secondary seed culture solution;

S4, fermenting, namely inoculating the secondary seed culture solution into a fermentation tank according to an inoculum size of 1% -5%, adding a feed medium for feeding, adding IPTG for induction expression when the bacterial density of the bacterial reaches 30% -40, performing tank unloading operation when the OD value reaches above 70, collecting fermentation liquor, centrifuging, and collecting the bacterial;

S5, purifying in the first step, namely re-suspending, crushing and centrifuging the bacteria, collecting supernatant, subjecting the supernatant to Ni column affinity chromatography, eluting, and collecting eluent;

S6, purifying the eluent in the second step through isoelectric point precipitation or Q anion exchange or SP ion exchange, and collecting supernatant or eluent to obtain the tetanus toxoid.

2. The method for producing tetanus toxoid according to claim 1, wherein in S1, the TeNT-Hc strain is an engineering bacterium comprising a tetanus toxin receptor binding region Hc nucleotide sequence.

3. The process for the preparation of tetanus toxoid according to claim 1, characterized in that in S2 the seed culture medium comprises 5g/L yeast powder, 10g/L peptone and 10g/L NaCl.

4. The method for preparing tetanus toxoid according to claim 1, wherein in the step S3, the further amplification of the strain number specifically comprises the steps of filling a seed culture medium into a shake flask for sterilization, cooling to room temperature, inoculating a primary seed culture solution into the seed culture medium according to an inoculation amount of 0.1% -0.5%, placing into a shaking table, and culturing for 3-8 hours at a constant temperature of 37 ℃ until an OD ₆₀₀ value reaches 0.8-1.5, thus obtaining a secondary seed culture solution.

5. The process for the preparation of tetanus toxoid according to claim 1, characterized in that in S4 it comprises in particular:

s41, providing a fermentation tank, carrying out constant volume according to a fermentation medium formula, and sterilizing for 15-25 min at 110-130 ℃;

s42, inoculating the secondary seeds into a fermentation tank according to an inoculum size of 1% -5%, controlling the temperature to be 37℃, pH and 7.0, controlling the dissolved oxygen to be more than or equal to 30%, and adding a feed medium to feed when the dissolved oxygen rises to more than 60% rapidly, wherein the initial feed speed is 15 g/(L.times.h), and controlling the dissolved oxygen to be more than or equal to 30% after feeding;

s43, when the bacterial density reaches 30-40, starting to cool, wherein the temperature is set to be 16-25 ℃;

S44, adding 0.1 mM-1 mM MIPTG for induction for 8-12 h;

s45, when the OD value reaches more than 70, carrying out tank discharging operation, and collecting fermentation liquor;

S46, centrifuging the fermentation liquor, and collecting the centrifuged thalli.

6. The process for preparing tetanus toxoid according to claim 1, wherein in S4, the fermentation medium comprises 5g/L yeast extract, 5g/L ammonium sulfate, 10g/L glucose, 3.8g/L dipotassium hydrogen phosphate, 3.5g/L anhydrous potassium dihydrogen phosphate, 1g/L magnesium sulfate heptahydrate, 1g/L trace elements, 0.075g/L calcium chloride, and VB10.0022g/L.

7. The process for the preparation of tetanus toxoid according to claim 1, characterized in that in S4 the feed medium comprises 500g/L glucose, 20g/L magnesium sulphate and 1g/L trace elements.

8. The process for preparing tetanus toxoid according to claim 6 or 7, characterized in that in S4 the microelements comprise 5g/L NaCl, 4g/L MnCl ₂·4H₂ O, 1g/L ZnSO ₄·7H₂ O,0.5g/L sodium molybdate dihydrate, 4.75g/L FeCl ₃·6H₂ O, 0.575g/L boric acid, 0.4g/L cuso4.5h ₂ O, and 2.038ml/L concentrated sulfuric acid.

9. Process for the preparation of tetanus toxoid according to claim 1, characterized in that in S4, in S5, it comprises in particular:

s51, re-suspending the thalli according to 1:5 (M/V) and 20mM Tris-HCl (pH 7.5) buffer solution, crushing the thalli in a homogenizer at a pressure of more than 70MPa, and circulating the thalli for 20min;

S52, centrifuging the crushed liquid at a centrifugal force of 12000xg and a temperature of 4 ℃ for 20min to collect supernatant;

S53, purifying the supernatant by Ni column affinity chromatography on a Ni column after passing through a 1mM filter membrane, balancing with 20mM Tris-HCl (pH 7.5) buffer solution, washing impurities with 40mM imidazole, eluting with 500mM imidazole, and collecting eluent.

10. A tetanus toxoid produced by the method of any one of claims 1 to 9.