Recombinant tetanus toxin protein rTT as well as preparation method and application thereof

Abstract

The invention discloses a recombinant tetanus toxin protein rTT as well as a preparation method and application thereof, the recombinant protein sequentially contains a His tag, a modified exogenous polypeptide and two tetanus toxin fragments connected in series through flexible polypeptides from the N end to the C end, the two tetanus toxin fragments connected in series form two intramolecular disulfide bonds in a molecule through double mutation, and the tetanus toxin fragments are connected in series through flexible polypeptides. Coding nucleotide of the recombinant protein is expressed by escherichia coli BL21 (DE3), induced at low temperature and purified, and target protein with the purity larger than or equal to 95% can be obtained. A subunit vaccine prepared from the protein and a two-way oil adjuvant ISA201 enables mice to achieve 100% protection under 100 LD50 challenge dose, the protection effect is better than that of truncated tetanus toxin heavy chain fragment TTc protein, and the recombinant protein can be used for tetanus prevention vaccines and serological diagnostic reagents.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products, specifically relating to a recombinant tetanus toxin protein rTT, its preparation method, and its application. Background Technology

[0002] Bovine tetanus, also known as lockjaw or tetanus, is a toxic infectious disease caused by Clostridium tetani infection. It is characterized by muscle rigidity and involuntary excitation of the nervous system. Clinically, it often occurs in individuals, and cattle with deep or large wounds on their bodies are most susceptible to the disease. If infected cattle do not receive timely treatment, they are likely to die from the spread of tetanus toxin (TT).

[0003] The classic tetanus vaccine is prepared by inactivating tetanus toxin (TT) produced by Clostridium tetani with formaldehyde to obtain non-toxic TT. Clostridium tetani can form highly resistant spores, and TT is highly toxic. The preparation process is relatively strict, time-consuming, and costly. At the same time, the formaldehyde treatment of toxoids can easily cause environmental pollution. Therefore, there is room for further improvement in the existing process.

[0004] Expressing recombinant, non-toxic toxin proteins or fragments using genetic engineering techniques can eliminate the need for complex inactivation processes while preserving the immunogenicity of the toxin protein. Recombinant subunit vaccines could thus become a safer and more effective alternative vaccine. Tetanus toxin binds to gangliosides via two carbohydrate-binding sites on its C-terminal β-cloverleaf domain. It is internalized and retrogradely transported in motor neurons via the endogenous microtubule axonal pathway, reuptaken by the presynaptic membrane, and the L-chain is released into the cytoplasm. This L-chain acts on the Gln and Phe sites in vesicle-associated membrane protein 2 (WAMP2), preventing the N-ethylcis-succinimide fusion protein (NSF) from binding to WAMP2 to form the SNARE complex, thus inhibiting exocytosis. Due to the lack of the inhibitory substrate, the motor neurons are in a state of sustained excitation, leading to continuous muscle contraction and the development of tetanus symptoms. Therefore, TTc, as a non-toxic fragment of tetanus toxin, is an important candidate molecule for developing bovine tetanus recombinant subunit vaccines, serving as a protective antigen against tetanus.

[0005] Recombinant subunit vaccines are widely used due to their high safety profile, but TTc protein is prone to forming inactive inclusion body proteins in heterologous expression systems, resulting in relatively poor immunogenicity. Some studies have proposed adding large molecular weight solubilizing tags or co-expressing with molecular chaperones to achieve soluble expression of TTc protein, but these methods are difficult to mass-produce, limiting their application in veterinary vaccines. Summary of the Invention

[0006] To construct a highly efficient bovine tetanus severe subunit vaccine, this invention designs a recombinant tetanus toxin protein, which has good solubility and immunogenicity.

[0007] The present invention achieves the above objectives through the following technical solutions: First, a recombinant tetanus toxin protein was proposed, whose amino acid sequence includes a His tag, an exogenous polypeptide, and an amino acid fragment of the tetanus toxin TT protein. Its expression structure is shown below. Figure 1 As shown.

[0008] The His tag amino acid sequence is shown in SEQ ID NO:1, and this sequence is used for the purification of recombinant tetanus toxin protein; the exogenous polypeptide amino acid sequence is shown in SEQ ID NO:2, and this exogenous polypeptide sequence is derived from the N-terminal sequence of maltose-binding protein and is used to promote the soluble expression of recombinant tetanus toxin protein; the amino acid sequence of the tetanus toxin TT protein fragment is shown in SEQ ID NO:2. As shown in NO:3, to enhance the immunogenicity of recombinant tetanus toxin protein, tetanus toxin TT protein fragment 1 (830-1304 aa) and tetanus toxin TT protein fragment 2 (1117-1315 aa) were tandemly linked using the GGGGSGGGGSG method to form a single-chain dimer. This single-chain dimer protein contains two β-cloverleaf domains, with fragments 1 and 2 each containing one β-cloverleaf domain (1114-1300 aa). This β-cloverleaf domain contains a W-pocket lactose binding site and an R-pocket sialic acid binding site, which are important antigenic targets of the tetanus toxin TT protein. To further improve the stability of the recombinant tetanus toxin protein, the two tetanus toxin protein fragments were double-mutated: fragment 1 (G... 1160 C, N 1204 C), Fragment 2 (S) 1136 C, E 1185 C), thus forming two intramolecular disulfide bonds C 1160 —C 1185 C 1204 -C 1136 .

[0009] Then, a genetically engineered strain expressing recombinant tetanus toxin protein was constructed. Specifically, the recombinant tetanus toxin protein expression plasmid pET28a-rTT synthesized above was transformed into Escherichia coli BL21(DE3) competent cells, and after culture and sequencing, the recombinant expression strain BL21-rTT was obtained.

[0010] Finally, the recombinant expression strain BL21-rTT was cultured on a scale-up basis to express the recombinant tetanus toxin protein rTT, and then identified and purified. SDS-PAGE and Western blot analysis results showed that the recombinant expression strain BL21-rTT could efficiently and solublely express the recombinant tetanus toxin protein rTT. Ni-NTA affinity chromatography was used to achieve efficient purification of the recombinant tetanus toxin protein rTT.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The tetanus toxin protein rTT proposed in this invention is soluble and has good immunogenicity, avoiding the problem that TTc protein easily forms inactive inclusion body proteins in heterologous expression systems, resulting in relatively poor immunogenicity, thus improving its application in veterinary vaccines.

[0012] 2. Traditional tetanus toxoid vaccines have contributed to the development of the aquaculture industry, but they are prepared by chemically inactivating Clostridium tetani toxoid, resulting in a complex and crude production process. The vaccines contain various Clostridium tetani components, metabolites, and culture media, and the quality of toxoid varies significantly from batch to batch. Furthermore, tetanus toxoid vaccination may cause clinical reactions, leading to unnecessary side effects (mild: such as nausea and vomiting; severe: severe allergic reactions, such as difficulty breathing and local rashes). In contrast, vaccines based on the tetanus toxoid protein rTT are prepared through high-density fermentation expression and purification using a stable and efficient E. coli expression system. This process is simple, safe, provides good protection, exhibits minimal batch-to-batch variation, and demonstrates stable properties. Attached Figure Description

[0013] Figure 1 To determine the expression structure of recombinant tetanus toxin protein rTT; Figure 2 Map of the recombinant tetanus toxin protein expression vector; Figure 3 For the expression detection of recombinant expression strain BL21-rTT, where M: protein marker; 1: whole bacterial cells; 2: sonicated supernatant; 3: sonicated precipitate; Figure 4 To identify the expression of the recombinant expression strain BL21-rTT, where M: protein marker; 1: sonication supernatant; Figure 5 Purification and detection of recombinant tetanus toxin protein rTT, where M: protein marker; 1: sonication supernatant; 2: flow-through sample; 3-5: elution peak samples; Figure 6SDS-PAGE and Western blot assays were performed on recombinant tetanus toxin protein rTT. In each figure, A represents SDS-PAGE of recombinant tetanus toxin protein rTT, and B represents Western blot assay of recombinant tetanus toxin protein rTT. In each figure, M represents the protein marker; 1 represents recombinant tetanus toxin protein rTT; and 2 represents the tetanus toxin heavy chain fragment TTc. Figure 7 The results are from the detection of specific IgG antibodies in the serum of immunized mice. Figure 8 Analysis of serum IL-4 and IFN-γ cytokine levels in mice; Figure 9 The results show the survival rates of mice in different immunized groups after challenge with the virus. Detailed Implementation

[0014] The present application will be further described in detail below with reference to the embodiments.

[0015] Example 1: Synthesis of Recombinant Expression Vector 1. Gene synthesis and sequence design.

[0016] A recombinant tetanus toxin protein, the amino acid sequence of which comprises the following three parts: a His tag, an exogenous polypeptide component, and an amino acid fragment of the tetanus toxin TT protein, and its expression structure is shown below. Figure 1 As shown.

[0017] The His tag is located at the N-terminus, and the amino acid sequence is shown in SEQ ID NO:1. This sequence is used for the purification of recombinant tetanus toxin protein.

[0018] The amino acid sequence of the exogenous polypeptide is shown in SEQ ID NO:2. This exogenous polypeptide sequence was derived from the N-terminal sequence (26-50 aa) of maltose-binding protein and is used to promote the soluble expression of recombinant tetanus toxin protein. The amino acid sequence of the wild-type exogenous polypeptide is shown in SEQ ID NO:6.

[0019] The amino acid sequence of the tetanus toxin TT protein fragment is shown in SEQ ID NO:3. To improve the immunogenicity of the recombinant tetanus toxin protein, two key functional fragments of tetanus toxin (TT) were selected.

[0020] Tetanus toxin TT protein fragment 1 consists of amino acids 830 to 1304; tetanus toxin TT protein fragment 2 consists of amino acids 1117 to 1315; both fragment 1 and fragment 2 contain a β-cloverwise domain (1114-1300 aa), which has a W-pocket lactose binding site and an R-pocket sialic acid binding site. The two fragments are tandemly linked by GGGGSGGGGSG to form a single-chain dimer protein, thus containing two β-cloverwise domains.

[0021] To further improve the stability of the recombinant tetanus toxin protein structure, the two tetanus toxin protein (TT) fragments were double-mutated: fragment 1 was G. 1160 C (replace glycine (Gly, G) at position 1160 with cysteine ​​(Cys, C)), N 1204 C (replace asparagine (Asn, N) at position 1204 with cysteine ​​(Cys, C)); S in fragment 2. 1136 C (replace serine at position 1136 (Ser, S) with cysteine ​​(Cys, C)), E 1185 C (replacing glutamic acid (Glu, E) at position 1185 with cysteine ​​(Cys, C)) thus forming two intramolecular disulfide bonds C 1160 -C 1185 C 1204 -C 1136 This disulfide bond stabilizes the spatial positions of the two TT fragments, thereby exposing effective antigenic epitopes and enhancing the structural stability of the recombinant protein.

[0022] 2. Construction of recombinant expression vectors.

[0023] The nucleotide sequence encoding the recombinant tetanus toxin protein was synthesized by Nanjing Genscript Biotech Co., Ltd. using codon optimization for E. coli, and cloned into the pET28a expression vector (cloning site NcoI / XhoI), named the recombinant tetanus toxin protein expression vector pET28a-rTT. The recombinant tetanus toxin protein expression vector map is shown below. Figure 2 As shown in SEQ ID NO:4, the nucleotide sequence of the recombinant tetanus toxin protein is shown in SEQ ID NO:5.

[0024] Example 2: Construction of a genetically engineered strain expressing recombinant tetanus toxin protein The recombinant tetanus toxin protein expression plasmid pET28a-rTT synthesized in Example 1 was transformed into Escherichia coli BL21(DE3) competent cells. Single clones were picked and cultured in LB liquid medium containing kanamycin at 37°C with shaking overnight. After PCR identification, the target DNA fragment was confirmed. The samples were then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The strain with the correct sequence was identified as a positive strain and named recombinant expression strain BL21-rTT. Glycerol was added to a final concentration of 15% and the strain was frozen at -80°C for later use.

[0025] Example 3: Expression and identification of recombinant tetanus toxin protein rTT The recombinant expression strain BL21-rTT obtained in Example 2 was inoculated into 10 mL of LB liquid medium containing kanamycin and cultured at 37°C with shaking. When OD 600 When the bacterial concentration was 0.6–0.8, IPTG solution with a final concentration of 0.5 mM was added, and the cells were incubated at 18°C ​​for 12–16 h. After the bacterial culture was completed, the cells were collected by centrifugation, and the cells were resuspended at a ratio of 10 mL PBS lysis buffer per gram of wet weight of cells, and then sonicated. The disrupted bacterial culture was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected for later use. The whole bacterial cells, sonicated supernatant, and sonicated precipitate were subjected to 12% SDS-PAGE electrophoresis, and the supernatant protein sample was identified by Western blot using a His-tagged antibody.

[0026] SDS-PAGE test results are as follows: Figure 3 As shown, the recombinant expression strain BL21-rTT exhibited a distinct target protein band after induction, with a molecular weight of approximately 78 kDa, consistent with the theoretically expected size. Furthermore, the majority of the expressed target protein was present in the sonicated supernatant, with a small amount remaining in the precipitate. Figure 3 ImageJ analysis showed that the target protein accounted for 35% of the total soluble protein.

[0027] Western blot identification results are as follows: Figure 4 As shown, a distinct specific band also appeared in the supernatant protein, consistent with the SDS-PAGE detection results; the SDS-PAGE and Western blot detection results indicate that the recombinant expression strain BL21-rTT can efficiently and solublely express the recombinant tetanus toxin protein rTT.

[0028] Example 4: Purification of recombinant tetanus toxin protein rTT 1. Culture, collection, and disruption of bacterial cells The recombinant expression strain BL21-rTT, which was detected as expressed in Example 3, was inoculated into 400 mL of LB liquid medium containing kanamycin and cultured with shaking at 37°C. When OD... 600When the concentration is 0.6–0.8, IPTG solution with a final concentration of 0.5 mM is added and the mixture is incubated at 18°C ​​for 12–16 h.

[0029] After bacterial induction, the bacterial cells were collected by centrifugation. The cells were resuspended at a ratio of 10 mL PBS lysis buffer per gram of wet bacterial weight and homogenized using high pressure. The homogenized bacterial solution was then centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected and filtered through a 0.45 µm filter membrane for later use.

[0030] 2. Protein purification (1) Column pretreatment: use ultrapure water for pump cleaning at a flow rate of 5 mL / mL; install Ni-NTA column (purchased from Wuhan Huiyan Biotechnology Co., Ltd.), wash 5 column volumes with ultrapure water at a flow rate of 2 mL / min, and wash 5 column volumes with PBS equilibration buffer.

[0031] (2) Loading, washing and elution: Load 40 mL of cell lysate supernatant at a flow rate of 1 mL / min and collect the flow-through; wash with PBS containing 30 mM imidazole for 5 column volumes at a flow rate of 1 mL / min; elute with PBS containing 500 mM imidazole at a flow rate of 1 mL / min and collect the elution peak.

[0032] (3) Column preservation: Wash with 5 column volumes of ultrapure water, then wash with 5 column volumes of 20% ethanol at a flow rate of 2 mL / min. Store the column in a refrigerator at 4°C.

[0033] 3. Protein detection The purification effect was detected by 12% SDS-PAGE electrophoresis.

[0034] Purification results are as follows Figure 5 As shown, the presence of the target band in the ultrasound supernatant indicates good protein soluble expression; almost no recombinant tetanus toxin protein (rTT) band was found in the flow-through solution, indicating that recombinant tetanus toxin protein (rTT) can bind efficiently to the Ni-NTA column; while a clear recombinant tetanus toxin protein (rTT) band appeared in the elution solution, indicating that recombinant tetanus toxin protein (rTT) was eluted efficiently. Figure 5 Channels 3, 4, and 5 are the three elution peaks collected sequentially during the 500 mM imidazole elution process in step (2).

[0035] Therefore, Ni-NTA affinity chromatography can be used to achieve efficient purification of recombinant tetanus toxin protein rTT. After purification, the purity of the target protein was ≥95% as analyzed by ImageJ.

[0036] Example 5: Western blot detection of recombinant tetanus toxin protein rTT The recombinant tetanus toxin protein rTT purified in Example 4 and the tetanus toxin heavy chain fragment TTc previously expressed and purified in the laboratory were subjected to 12% SDS-PAGE electrophoresis and detected by Western blot using tetanus toxin-positive serum.

[0037] The tetanus toxin heavy chain fragment TTc, previously expressed and purified in the laboratory, contains only the receptor-binding domain of the heavy chain and is non-toxic, but retains most of the neutralizing antibody epitopes. Therefore, it is often used as a vaccine control antigen or an ELISA coating antigen. Using antigen TTc as a positive control demonstrates the effectiveness of the experimental system (serum, antibody, colorimetric assay).

[0038] The results are as follows Figure 6 As shown, the purified recombinant tetanus toxin protein rTT and tetanus toxin heavy chain fragment TTc can both be specifically recognized by tetanus toxin positive serum, indicating that the purified recombinant tetanus toxin protein rTT in Example 4 has good immunogenicity.

[0039] Example 6: Mouse Immunization Test Thirty 6-8 week old SPF-grade BALB / c female mice were divided into three groups of 10 mice each: negative control (PBS control group), rTT subunit vaccine group, and TTc subunit vaccine group.

[0040] The rTT and TTc subunit vaccines were prepared by emulsifying diluted rTT and TTc proteins with ISA201 adjuvant at a ratio of 1:1 (V:V), respectively, with an antigen content of 200 μg / mL for both.

[0041] All experimental mice were immunized subcutaneously, and a second immunization was performed 14 days after the first immunization. Each mouse in the negative control group was immunized with 100 μL of PBS, each mouse in the rTT subunit vaccine group was immunized with 100 μL of rTT unit vaccine, and each mouse in the TTc subunit vaccine group was immunized with 100 μL of TTc subunit vaccine.

[0042] Blood samples were collected from the orbital rims of all experimental mice before immunization, 14 days after the first immunization, and 14 days after the second immunization, and serum was separated for later use.

[0043] Example 7: ELISA detection of serum-specific IgG antibodies The antibody levels in the serum of mice in different immunization groups in Example 6 were determined using an enzyme-linked immunosorbent assay (ELISA). The specific procedure is as follows: (1) Antigen incubation. Tetanus toxin TT (purchased from Jilin Wuxing Animal Health Co., Ltd.) was diluted to 1 μg / mL, and 100 μL was added to each well of the ELISA plate. The plate was incubated overnight at 4°C. The next day, 200 μL of 2% skim milk blocking buffer was added to each well, and the plate was incubated at 37°C for 2 hours. The plate was then washed three times with PBST to allow the antigen to be adsorbed onto the plate.

[0044] (2) Add the antibody to be tested (primary antibody) to allow serum IgG to bind to the antigen from the previous step.

[0045] For the sample group, mouse serum samples were diluted with antibody diluent at gradients of 100x, 200x, 400x, 800x, etc. Then, 100 μL of the diluted serum to be tested was added to each well of the ELISA plate.

[0046] Positive control group: High-titer standard serum, at the same dilution, repeated in 2 wells; Negative control: Serum from unimmunized mice, at the same dilution, replicated in 2 wells. The perforated plate was incubated in a 37°C incubator for another 1.5 hours.

[0047] (3) Add secondary antibody. After washing 3 times with PBST, add 100 μl of horseradish enzyme-labeled goat anti-mouse IgG diluted 1:5000 to each well as secondary antibody. React in a constant temperature incubator at 37℃ for 0.5 h, and wash 3 times with PBST.

[0048] (4) Color development and termination: Add 50 μL of substrate solution to each well and develop color in the dark for 15 min. After color development, add 50 μL of stop solution to each well to terminate the reaction. Immediately read the results at 450 nm wavelength using an ELISA reader.

[0049] Mean OD of positive control serum 450 ≥1.0, mean OD of negative control serum 450 The result is valid if the absorbance of the experimental group is ≤0.3 compared to the negative control group. A positive result is defined as an absorbance value ≥2.1 in the experimental group compared to the negative control group, and a negative result is defined as an absorbance value ≤1.5 in the experimental group compared to the negative control group.

[0050] Indirect ELISA results as follows Figure 7 As shown, 14 days after the first immunization, the IgG antibody titers of all mice in the rTT immunization group were no less than 1:25600, and the IgG antibody titers of all mice in the TTc immunization group were no less than 1:6400, indicating strong IgG antibody titer levels. 28 days after the first immunization (14 days after the second immunization), the IgG antibody titers of all mice in the rTT immunization group were no less than 1:51200, and the IgG antibody titers of all mice in the TTc immunization group were no less than 1:12800. The results indicate that both the rTT and TTc immunization groups produced strong IgG antibody titers, with the IgG antibody titer in the rTT immunization group being significantly higher than that in the TTc immunization group.

[0051] Example 8 Cytokine Detection To assess the immune type and related cytokine levels secreted by T cells in mice after immunization, cytokine levels in serum from mice in different immunization groups at 0d, 14d, and 28d were measured using an IL-4 and IFN-γ cytokine assay kit (purchased from Beyotime Biotechnology Co., Ltd.). The specific procedures were performed according to the cytokine assay kit instructions.

[0052] The results are as follows Figure 8 As shown, the levels of IL-4 and IFN-γ cytokines induced by rTT and TTc immunization groups were significantly higher than those in the PBS control group, and the levels of IL-4 and IFN-γ cytokines induced by rTT immunization group were higher than those in the TTc immunization group. These results indicate that rTT induces both cellular and humoral immune responses, and the cellular and humoral immune responses induced by the rTT group are stronger than those induced by the TTc group.

[0053] Example 9 Mouse challenge experiment To further evaluate the protective efficacy of the rTT subunit vaccine, 21 days after the second immunization, all experimental mice (all mice in Example 6) were intraperitoneally injected with 500 μL of diluted TT (containing 100 L of TT). 50 , 1.5ng), and the survival of mice in each group was observed and recorded within 1 week after injection.

[0054] The results are as follows Figure 9 As shown, mice in the PBS control group began to die on day 1 after challenge, and all mice in the PBS control group died on day 3. One mouse in the TTc immunization group died on days 3 and 4 after challenge. On day 7 after challenge, the survival rates of mice in the PBS control group, rTT group, and TTc group were 0%, 100%, and 80%, respectively, and the survival rates of each experimental group tended to stabilize.

[0055] The results showed that the rTT subunit vaccine could completely protect mice from tetanus toxin attack, and the protective effect of the recombinant tetanus toxin protein rTT subunit vaccine was better than that of the tetanus toxin heavy chain fragment TTc subunit vaccine.

[0056] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A recombinant tetanus toxin protein, characterized in that, The protein contains, from N-terminus to C-terminus, a His tag, an exogenous polypeptide, tetanus toxin TT protein fragment 1, and tetanus toxin TT protein fragment 2. The exogenous polypeptide is a soluble peptide modified by multiple point mutations of the N-terminal 24 amino acids of maltose-binding protein. The tetanus toxin TT protein fragment 1 is amino acids 830-1304 of the full-length amino acid sequence of tetanus toxin and contains G1160C and N1204C mutations. The tetanus toxin TT protein fragment 2 is amino acids 1117-1315 of the full-length amino acid sequence of tetanus toxin, and contains S1136C and E1185C mutations. The tetanus toxin TT protein fragment 1 and tetanus toxin TT protein fragment 2 are linked by a flexible linker peptide GGGGSGGGGSG. The Cys1160 of fragment 1 and the Cys1185 of fragment 2 form a first intramolecular disulfide bond, and the Cys1204 of fragment 1 and the Cys1136 of fragment 2 form a second intramolecular disulfide bond, causing the protein to present a single-chain dimer conformation. The full-length amino acid sequence of tetanus toxin is shown in SEQ ID NO:

3.

2. The recombinant tetanus toxin protein according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

5.

3. A nucleic acid molecule encoding the recombinant tetanus toxin protein of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

4.

4. A vector for expressing recombinant tetanus toxin protein, characterized in that, The vector contains the nucleic acid molecule of claim 2, the nucleotide sequence of which is shown in SEQ ID NO:

4.

5. A genetically engineered strain, characterized in that, The strain is a derivative of Escherichia coli BL21(DE3), and the expression vector of claim 4 is integrated into the genome of the strain.

6. A method for preparing the recombinant tetanus toxin protein of claim 1, characterized in that, Includes the following steps: (1) Cultivation: The genetically engineered strain containing the vector of claim 4 or the genetically engineered strain containing the target expression vector of claim 5 is inoculated into kanamycin-resistant LB medium and amplified at 37 °C. (2) Induction: Add 0.5 mM IPTG to the bacterial culture above, cool to 18℃ and induce culture to achieve soluble expression of the target protein; (3) Break the bacteria. After the induction is complete, centrifuge to collect the bacterial cells, add PBS to resuspend them, and then use high pressure homogenization to break the bacteria completely. (4) Clarification: Centrifuge the lysate from the above steps to remove cell debris and insoluble matter. Filter the supernatant through a 0.45 μm filter membrane to obtain a clear lysate. (5) Purification: Add the clarified lysis buffer to the Ni-NTA affinity chromatography column to remove impurities first, and then elute in one step with 500 mM imidazole buffer and collect the elution peak. (6) Change the eluent and concentrate it in an ultrafiltration centrifuge tube. After repeated dilution and concentration, remove the imidazole buffer to obtain the purified recombinant tetanus toxin protein, which can be stored at 4°C for later use.

7. The method according to claim 6, characterized in that, The method further includes step (7) identification, in which the protein obtained in step (6) is identified using 12% SDS-PAGE and Western blot. In the Western blot, tetanus toxin-positive serum is used as the primary antibody. The appearance of the expected molecular weight band indicates that the target protein is correctly expressed and has immunoreactivity.

8. A subunit vaccine composition, characterized in that, Include: (a) The recombinant tetanus toxin protein according to any one of claims 1 to 2; and (b) A pharmaceutically acceptable adjuvant, wherein the adjuvant is the biphasic oil adjuvant ISA201.

9. The subunit vaccine composition according to claim 8, characterized in that, The composition is an intramuscular injection, and the content of recombinant tetanus toxin protein is 200 μg / ml.

10. The use of the recombinant tetanus toxin protein of claim 1 in the preparation of a vaccine for the prevention of tetanus or a diagnostic reagent for the detection of tetanus antibodies.

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