CN116270998A - Multivalent biotoxin antigen vaccine based on double receptor binding region RBD assembly and its preparation method and application - Google Patents

Abstract

The invention discloses a multivalent biotoxin antigen vaccine based on double receptor binding region RBD assembly, its preparation method and application. The invention provides a multivalent biotoxin molecular antigen vaccine, the active ingredient of which is a fusion protein formed by linking the Hc protective antigen of biotoxin 1 and the Hc protective antigen of biotoxin 2 through a connecting peptide; the biotoxin 1 and the biological toxin 2 are two different biological toxins; the biological toxins are selected from the following: tetanus toxin or other types of botulinum toxin other than type E (including types A, B and H, etc.). The present invention proves that the Hc antigens of different serotypes of botulinum toxin and tetanus toxin receptor binding region are fused and assembled into double Hc fusion antigen molecules by using genetic engineering and biosynthesis technology, which can produce a strong anti-multiple subunit vaccine as a single subunit vaccine. protection against biotoxins. Therefore, the Hc fusion antigen molecular vaccine of the present invention can produce protective effects against multiple different serotypes of biological toxin pathogens, and can be used as a broad-spectrum multivalent vaccine for the prevention and preventive treatment of biological toxins.

Description

Multivalent Biotoxin Antigen Vaccine Based on Double Receptor Binding Domain RBD Assembly and Its Preparation Preparation method and application

technical field

The invention relates to the field of biopharmaceuticals, in particular to a multivalent biotoxin antigen vaccine based on double receptor binding domain RBD assembly and its preparation method and application.

Background technique

Botulinum neurotoxin (BoNTs) is a neurotoxin secreted by Clostridium botulinum. It is the most toxic substance among biological and chemical poisons known to humans. The half-lethal dose LD ₅₀ for humans is only 0.1-1ng/kg . Botulinum toxin can be divided into eight serotypes (AH) according to its antigenic properties, among which botulinum toxin types A, B, E, F, and H can cause botulism in humans. H-type new botulinum toxin is a new serotype discovered by biologists in 2013. It is a hybrid of A and F types. Its Hc is similar to BoNT/A1, with 84% sequence homology, and can be anti-A type. Toxin antibodies were partially neutralized, and HN and L shared 64% and 81% homology with BoNT/F1 and BoNT/F5, respectively.

Because Clostridium botulinum, which produces botulinum toxin, exists widely in nature and its spores have strong resistance to the external environment, botulinum toxin poisoning is still a very serious public health problem. Therefore, it is of great practical significance to carry out the research and development of prevention and treatment drugs for botulinum toxin.

Tetanus toxin (TeNT) is a toxin produced by Clostridium tetani invading the human body through skin or mucous membrane wounds and growing and multiplying in a hypoxic environment. Tetanus toxicity is also very strong, the estimated lethal dose to human body is lower than 2.5ng/kg, and the untreated case fatality rate is as high as 40%. Tetanus is a life-threatening disease characterized by muscle spasms caused by the neurotoxin of Clostridium tetani. Often secondary to various traumas, it can also occur in mothers and newborns who give birth under unclean conditions.

Tetanus toxin has the same structural features and similar mechanism of intoxication as botulinum toxin. Mature active botulinum toxin and tetanus toxin are both long peptide chains consisting of a light chain (L chain, 50 kDa) and a heavy chain (H chain, 100 kDa) linked together by non-covalent disulfide bonds. The light chain is a toxic domain, which has zinc ion endopeptidase activity; the N-terminal of the heavy chain (HN domain), the transport region is mainly composed of α-helix, and plays an important role in the transmembrane transport of biotoxins; the heavy chain C The end (Hc domain) is a nerve cell-specific binding domain, consisting of two subdomains (Hc-N and Hc-C), interacting with receptor proteins and intervening in the entry of neurotoxins into cells.

Botulinum toxin is the most toxic protein known so far, and the development of its vaccine and neutralizing antibody has attracted much attention. However, the current research or limited use of toxoid vaccines have many shortcomings and cannot be popularized and applied. In recent years, the research on botulinum toxin vaccine has been strengthened at home and abroad, trying to find a safe and effective vaccine, and the most promising one is the new recombinant subunit vaccine. Most previous studies have shown that the botulinum toxin receptor binding domain (RBD) Hc is the basic determinant of the protective antigen, which has a complete protective effect and is a main target antigen for botulinum toxin vaccine research. At the same time, the tetanus toxin receptor binding region Hc is also an important protective antigen, which can induce the body to produce a strong protective effect. This tetanus recombinant subunit vaccine does not require the cultivation of tetanus bacteria or the purification of tetanus toxin, making the production process safer and more efficient, and has the potential to replace formaldehyde-inactivated tetanus toxin (toxoid molecule) as a new type of vaccine.

Multivalent multivalent vaccines are the main development trend of the vaccine industry at home and abroad. Relying on the three major engineering technologies of genes, fermentation, and proteins, multivalent multivalent vaccines are more effective in the screening and preparation of pathogenic protective antigens and efficient and stable preparation processes. Important progress has been made in key technologies. Multiple multivalent vaccines can protect multiple pathogens at the same time, especially one vaccine can protect multiple pathogens, which has significant advantages and application prospects. In view of the characteristics of botulinum toxin with multiple serotypes and multiple subtypes, as well as the complexity and diversity of its toxin-producing strains, as well as its diversity and variability, it is difficult for monovalent vaccines to protect against multiple biological toxins or multiple biological toxins simultaneously. poisoning. Therefore, for various types of botulinum toxin and tetanus toxin pathogens, it is of great significance and application prospect to study multivalent biotoxin antigen vaccines with the help of mature monovalent candidate vaccine protective antigens. This multivalent vaccine can protect multiple botulinum toxins at the same time. toxin or botulinum toxin and tetanus toxin.

Contents of the invention

The purpose of the present invention is to provide a multivalent biotoxin antigen vaccine based on double receptor binding domain RBD assembly and its preparation method and application.

In the first aspect, the present invention claims a polyvalent biological toxin molecular antigen vaccine.

In the present invention, the biological toxin is tetanus toxin or botulinum toxin.

The active ingredient of the multivalent biotoxin molecular antigen vaccine claimed in the present invention is a fusion protein formed by linking the Hc antigen of biotoxin 1 and the Hc antigen of biotoxin 2 through a connecting peptide;

The biotoxin 1 and the biotoxin 2 are two different biotoxins;

Said biological toxin is selected from the following: tetanus toxin or other types of botulinum toxin other than type E.

In a specific embodiment of the present invention, the other types of botulinum toxin other than type E are specifically botulinum toxin type A, botulinum toxin type B or botulinum toxin type H.

Further, the active ingredient of the polyvalent biotoxin molecular antigen vaccine is specifically any of the following:

(A1) A fusion protein formed by linking the Hc antigen of botulinum toxin type A and the Hc antigen of tetanus toxin through a linking peptide in the order from amino acid to carboxyl terminal, and the fusion protein is named AHc-THc;

(A2) A fusion protein formed by linking the Hc antigen of tetanus toxin and the Hc antigen of botulinum toxin through a linking peptide in the order from amino acid to carboxyl terminal, and the fusion protein is named THc-AHc;

(A3) A fusion protein formed by linking the Hc antigen of botulinum toxin type A and the Hc antigen of botulinum toxin type B through a linking peptide in the order from the amino acid to the carboxyl terminal, and the fusion protein is named AHc-BHc;

(A4) A fusion protein formed by linking the Hc antigen of botulinum toxin type B and the Hc antigen of botulinum toxin type H via a linker peptide in the order from the amino acid to the carboxyl terminal. The fusion protein is named BHc-HHc.

Furthermore, the connecting peptide is (G4S)3, and the specific amino acid sequence is: GGGGSGGGGSGGGGSGS.

Furthermore, the amino acid sequence of the Hc antigen of the botulinum toxin type A is shown in SEQ ID No.10.

Furthermore, the amino acid sequence of the Hc antigen of the botulinum toxin type B is shown in SEQ ID No.12.

Furthermore, the amino acid sequence of the Hc antigen of the botulinum toxin type H is shown in SEQ ID No.8.

Furthermore, the amino acid sequence of the Hc antigen of the tetanus toxin is shown in SEQ ID No.16.

More specifically, the amino acid sequence of the AHc-THc is shown in SEQ ID No.18.

More specifically, the amino acid sequence of THc-AHc is shown in SEQ ID No.20.

More specifically, the amino acid sequence of the AHc-BHc is shown in SEQ ID No.26.

More specifically, the amino acid sequence of the BHc-HHc is shown in SEQ ID No.28.

In the second aspect, the present invention claims a method for preparing the polyvalent biotoxin molecular antigen vaccine described in the first aspect above.

The method for preparing the polyvalent biotoxin molecule antigen vaccine described in the first aspect of the present invention may include the following steps: introducing the nucleic acid molecule encoding the fusion protein into E. coli recipient cells to obtain recombinant E. coli; The recombinant Escherichia coli is cultivated to obtain the fusion protein; and then the multivalent biotoxin molecular antigen vaccine is prepared by using the fusion protein as an active ingredient.

Further, the nucleic acid molecule sequence encoding the Hc antigen of botulinum toxin type A is shown in SEQ ID No.9.

Further, the nucleic acid molecule sequence encoding the Hc antigen of botulinum toxin type B is shown in SEQ ID No.11.

Further, the nucleic acid molecular sequence encoding the Hc antigen of the H-type botulinum toxin is shown in SEQ ID No.7.

Further, the nucleic acid molecule sequence encoding the Hc antigen of the tetanus toxin is shown in SEQ ID No.15.

Furthermore, the nucleic acid molecule sequence encoding the AHc-THc is shown in SEQ ID No.17.

Furthermore, the nucleic acid molecule sequence encoding THc-AHc is shown in SEQ ID No.19.

Furthermore, the nucleic acid molecule sequence encoding the AHc-BHc is shown in SEQ ID No.25.

Furthermore, the nucleic acid molecule sequence encoding the BHc-HHc is shown in SEQ ID No.27.

In the method, in the process of culturing the recombinant Escherichia coli, IPTG is added to the logarithmic growth phase to a final concentration of 0.4mmol/L, and then cultured at 30°C for 4-5h or overnight at 18°C Cultivation; after the cultivation, the bacterial cells are collected, ultrasonically disrupted, and the supernatant is collected by centrifugation, and the fusion protein can be obtained from the supernatant.

Further, after the supernatant is collected by centrifugation, a purification step is also included using a Ni-NTA affinity chromatography column or a streptavidin affinity chromatography column.

In the above two aspects, the multivalent biotoxin molecule antigen vaccine also contains an adjuvant in addition to the active ingredient.

Further, such as aluminum adjuvant.

In the third aspect, the present invention claims any of the following substances:

(B1) protein, which is the fusion protein described in the first aspect above;

(B2) a nucleic acid molecule, which is the nucleic acid molecule described in the first aspect above, encoding the protein described in (B1);

(B3) recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the nucleic acid molecules described in (B2);

(B4) A product for the prevention and/or treatment of poisoning symptoms caused by botulinum toxin and/or tetanus toxin, containing the protein described in (B1) or the nucleic acid molecule described in (B2) or the nucleic acid molecule described in (B3) The above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria.

In the fourth aspect, the present invention claims to protect the protein described in (B1) or the nucleic acid molecule described in (B2) or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria described in (B3) in the third aspect above. Use in a product for the prevention and/or treatment of intoxication conditions caused by botulinum toxin and/or tetanus toxin. Wherein, the botulinum toxin can also be type H botulinum toxin.

The applicant's team has established the cutting-edge foundation and immune efficacy research technology platform of each functional domain of botulinum toxin, and systematically completed the research on various types of botulinum toxin such as types A, B, E, F and H and functional epitopes of tetanus toxin The study of biological activity and immune efficacy has identified a series of new functional epitopes and immune molecules with strong protective effects, such as receptor binding region A/B-Hc or light chain-transmembrane region E/F-L-HN, etc. , providing a wealth of candidate protective antigen molecules for the development of broad-spectrum high-efficiency multivalent botulinum toxin vaccines. In addition, the present invention has explored the immune efficacy of each functional domain epitope antigen of H-type botulinum toxin, and the results show that the H-type receptor binding region HHc also has a strong protective effect, and it has a strong protective effect with the A-type botulinum toxin receptor binding region AHc has a strong cross-protective effect. Since botulinum toxin and tetanus toxin receptor binding domain Hc (Receptor binding domain, RBD) are important protective antigens, they can be used as subunit candidate vaccines. The technical solution is to use recombinant technology to fuse and assemble Hc antigens in the receptor binding region of different serotypes of botulinum toxin or tetanus toxin into a double Hc antigen molecular structure, and prepare a double Hc fusion antigen molecule through biosynthesis. The double Hc fusion antigen molecule As a polyvalent biotoxin vaccine, it is used to prevent multiple biotoxin pathogens.

Based on this concept and technology, the present invention explores a series of double Hc (or double receptor binding domain RBD) fusion antigen molecules, and verifies the feasibility of this technical solution and the effectiveness of the protective effect of multivalent vaccine antigen molecules. For example, AHc-THc and THc-AHc antigen molecules were prepared by combining the fusion molecular structure of type A botulinum toxin and tetanus toxin. Strong protective effect of botulinum toxin type H and tetanus toxin, and it can also cross-protect new type H botulinum toxin. Similarly, other type combinations such as AHc-BHc and BHc-HHc can also produce strong protective effects against botulinum toxins such as types A and B. The above results show that the single fusion antigen molecular vaccine can produce protective effects against two or three different serotype toxin pathogens, and can be used as a broad-spectrum multivalent vaccine for the prevention and preventive treatment of biological toxins. The above results show that the technology of multivalent biotoxin antigen assembled based on double receptor binding region RBD is feasible and can be applied to the research and development of multivalent vaccines against various biotoxin pathogens.

Description of drawings

Figure 1 is the SDS-PAGE electrophoresis and Western Blot identification diagrams of the purified H-type botulinum toxin functional domains HL, HHc, HHN, and HL-HN proteins. A is the SDS-PAGE identification of each functional domain protein of H-type botulinum toxin and other types of protein antigens, and lanes 1-7 are FHc, AHc, FL-HN, HL, HHc, HHN and HL-HN protein samples, respectively. Wherein FHc means F-type botulinum toxin Hc; FL-HN means F-type botulinum toxin L-HN; AHc means A-type botulinum toxin Hc, and they are all used as control functional domain proteins. B is the Western blot identification of various types of botulinum toxin receptor binding region protein antigens (the primary antibody test result is the hyperimmune serum against H type botulinum antitoxin receptor binding region), and lanes 1-3 are HHc, AHc, FHc. C is the Western blot identification of various types of botulinum toxin receptor binding region protein antigens (horse-derived hyperimmune serum against type A botulinum antitoxin receptor binding region is the test result of the primary antibody), and swimming lanes 1-3 are HHc, AHc, FHc. D is the Western blot identification of H-type botulinum toxin L and HN functional binding regions and FL-HN protein antigen (the result of the primary antibody test using mouse-derived hyperimmune serum against F-type botulinum toxin FL-HN), lane 1- 4 are HL, HN, HL-HN, FL-HN respectively. E is the Western blot identification of H-type botulinum toxin L and HN functional binding regions and FL-HN protein antigen (the primary antibody test result is the mouse-derived anti-H-type botulinum toxin HL-HN hyperimmune serum), swimming lane 1- 4 are HL, HN, HL-HN, FL-HN respectively. The box is the target protein. M is protein marker (from top to bottom: 170, 130, 90, 70, 55, 40, 35, 25, 15 and 10kDa).

Fig. 2 is a schematic diagram of the structure of a multivalent biotoxin antigen vaccine molecule assembled based on a double receptor binding domain RBD. Remarks: The selected receptor binding regions Hc and assembly sequence of various types of botulinum toxin and tetanus toxin are shown in the figure. The numbers in brackets indicate the amino acid sequence information of the receptor binding regions Hc, and the two receptor binding regions Hc- The RBD is assembled into a dual receptor-binding domain Hc molecule via a (G4S) _3- linked peptide. GS linker shows (G4S) ₃ linker peptide.

Figure 3 is the SDS-PAGE electrophoresis and Western Blot identification diagrams of THc-linker-AHc, AHc-linker-THc, THc, and AHc antigenic proteins obtained through purification after biosynthesis. Swimming lanes 1-4 are respectively THc-linker-AHc (100kDa), AHc-linker-THc (100kDa), THc (50kDa), AHc (50kDa) protein samples; M is protein marker (from top to bottom: 170, 130, 90, 70, 55, 40 and 35 kDa). A is the fusion receptor binding region of tetanus toxin and botulinum toxin type A THc-linker-AHc antigen, AHc-linker-THc antigen, separate tetanus toxin receptor binding region THc antigen and botulinum toxin type A receptor SDS-PAGE identification of binding region AHc antigen. B is the Western blot identification of the fusion receptor binding region of tetanus toxin and botulinum toxin type A and the antigen of the single receptor binding region (the standard substance of horse-derived botulinum toxin type A antitoxin is the test result of the primary antibody). C is the Western blot identification of the fusion receptor binding region of tetanus toxin and botulinum toxin type A and the individual receptor binding region antigen (the detection result of the primary antibody is the hyperimmune serum of mouse anti-tetanus toxin receptor binding region ).

Fig. 4 is the SDS-PAGE electrophoresis identification chart of recombinant AHc-linker-EHc, EHc-linker-AHc, AHc-linker-BHc, BHc-linker-HHc proteins obtained by purification after biosynthesis. In A, lane 1 shows the control strain that does not express the target protein; lane 2 shows the strain that biosynthesized and expressed the EHc-linker-AHc protein; lane 3 shows the purified EHc-linker-AHc protein (100kDa); lane 4 shows A strain expressing AHc-linker-EHc protein was biosynthesized; Lane 5 shows the purified AHc-linker-EHc protein (100kDa). In B, lane 1 shows the control strain that does not express the target protein; lane 2 shows the strain that biosynthesized and expressed the AHc-linker-BHc protein; lane 3 shows the purified AHc-linker-BHc protein (100kDa); lane 4 shows A strain expressing BHc-linker-HHc protein was biosynthesized; Lane 5 shows the purified BHc-linker-HHc protein (100 kDa).

Fig. 5 shows the results of antibody levels of each functional domain HL, HHc, HHN, and HL-HN protein antigens of type H botulinum toxin after immunizing mice. A is the antibody titers of HL, HHN, HL-HN and HHc antigen-immune sera against their respective antigens; B is the anti-AHc and anti-FL-HN antibody titers of HHc and HL-HN antigen-immune sera.

Fig. 6 shows the result of the antibody level after combined immunization of mice with tetanus toxin and type A botulinum toxin double receptor binding region Hc fusion molecule and separate receptor binding region functional epitope antigen protein. A is recombinant antigen THc-linker-AHc, AHc-linker-THc immunization 1 μg and 4 μg group for 1 time and 2 times immunization, THc+AHc combined with 1 μg group immunization 1 time and immunization mice for 2 times against each recombinant antigen in serum ( Antibody titers of THc-linker-AHc, AHc-linker-THc, AHc+THc). B is the recombinant antigen THc-linker-AHc, AHc-linker-THc 1 and 2 times immunization 1μg and 4μg groups, THc+AHc combined with 1μg group 1 and 2 times immunization of mice serum anti-AHc antibodies Titer. C is the recombinant antigen THc-linker-AHc, AHc-linker-THc 1 and 2 times immunization 1μg and 4μg groups, THc+AHc combined with 1μg group 1st immunization and 2 times immunization of mice serum anti-THc antibodies Titer.

Note: All the data in this experiment were analyzed by GraphPad Prism 5.0, and the experimental results were used as mean ± standard error (mean ± SD), and the statistical significance of the differences between paired experiments was analyzed by t-test or chi-square test.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be found in: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3 ^rd edition, 2001, NY, Cold Spring Harbor) and "The Pharmacopoeia of the People's Republic of China" (edited by the National Pharmacopoeia Committee, three volumes in 2020, Chemical Industry Press).

The following examples explored for the first time the immune efficacy of each functional domain epitope antigen of H-type botulinum toxin, and the results showed that the H-type receptor binding region HHc also has a strong protective effect, and it has a strong protective effect with the A-type botulinum toxin receptor The binding domain AHc has a strong cross-protection effect. Then a series of double Hc fusion antigen molecules were designed and prepared, and their structural properties and immunological activity were systematically evaluated, and the protective efficacy of single fusion antigen molecule vaccines against a variety of different toxin pathogens was evaluated. The purpose of this technical solution is to use recombinant technology to fuse the RBD antigen proteins of different serotype biotoxin receptor binding regions into a double Hc antigen molecular structure, assemble and prepare a double Hc fusion antigen molecule, and the double Hc fusion antigen molecule is used as a multivalent biological Toxin vaccines for protection against a variety of toxin biological agents.

Example 1, Expression, Purification and Identification of Recombinant Proteins of Recombinant Proteins with Epitopes of Functional Domains of Type H Botulinum Toxin in Escherichia coli Hc) Gene Design and Synthesis

According to the codon degeneracy, the recombinant protein genes encoding the functional epitopes of H-type botulinum toxin were artificially optimized and synthesized, and first cloned directly into cloning vectors such as pMD18-T (TaKaRa) vectors, respectively encoding HL, HHN, and L-HN and HHc (see Table 1 for detailed recombinant protein and sequence information). When cloning these artificially synthesized genes, in order to facilitate the following operations, the 5' end of each functional epitope antigen gene was introduced into EcoR I and the 3' end introduced the Xho I restriction recognition site.

The above gene design adopts codons commonly used in Escherichia coli, and ensures that the encoded amino acid residue sequence remains unchanged. According to the full-length gene sequence and amino acid residue sequence of botulinum toxin type H (BoNT/H, strain WP_047402807.1, full-length 1288 amino acids), all functional epitope antigen genes are optimized, which is more conducive to gene expression.

Table 1. Basic information table of epitope antigens of each functional domain of botulinum toxin type H

2. Construction of each recombinant prokaryotic expression vector

Each plasmid obtained above was double-digested with EcoR I and Xho I respectively, and the corresponding target gene fragments were respectively recovered with a DNA recovery kit, and the prokaryotic expression vector pTIG-Trx (see patent: ZL200710089588.2 ) connection, transform the ligation product into Escherichia coli (E.coli) DH5α competent cells, screen positive clones, extract plasmids, sequence, obtain recombinant prokaryotic expression vectors with correct sequence and insertion position, and name them pTIG according to the difference of the inserted fragments - Trx-HL, pTIG-Trx-HHN, pTIG-Trx-HL-HN and pTIG-Trx-HHc.

3. Expression of recombinant proteins in Escherichia coli and purification and identification of expression products

1. Expression of each recombinant protein in Escherichia coli and SDS-PAGE detection of the expression product

The 4 recombinant prokaryotic expression vectors constructed in step 2 were transformed into Escherichia coli BL21 (DE3) competent cells (TIANGEN Company) respectively, and positive recombinants were screened, and the recombinant bacteria transformed with pTIG-Trx empty vector were used as negative controls, and then pressed 1 Inoculate the positive recombinant bacteria into 500mL LB liquid medium containing 100mg/mL ampicillin at a ratio of 100, and carry out mass culture at 37°C and 250rpm until the logarithmic growth phase (OD ₆₀₀ is about 0.6-0.8) Add the chemical inducer IPTG to a final concentration of 0.4mmol/L, and culture with shaking at 220r/min at 18°C overnight or 4-5 hours at 30°C with shaking at 220r/min. After the culture, the bacteria were collected by centrifugation, resuspended in 20mM sodium phosphate buffer (pH 8.0), the cells were broken by ultrasonic, and the supernatant was collected by centrifugation for 12% SDS-PAGE detection. The results showed that all the induced recombinant proteins were It has been expressed and can exist in a soluble form, but the target protein band does not appear in the uninduced strain and the induced empty vector control, indicating that the expressed protein may be the target protein, that is, each recombinant protein.

2. Purification and identification of expression products

The C-terminus of each recombinant protein expressed in step 1 contains six histidine tags, so the soluble expression product is purified with Ni-NTA affinity chromatography column (Pharmacia) and referring to the instructions to obtain the eluted purified protein , and then the purified protein was detected by 12% SDS-PAGE, and the detection result showed that the purified target protein was obtained. The obtained target protein was stored at -20°C or -80°C for later use. Shown in A in Figure 1 is the SDS-PAGE electrophoresis of the four target proteins after purification.

3. Western blot identification of expression products

Use mouse-derived anti-H-type botulinum toxin or anti-A and F-type botulinum toxin serum antibodies (hyperimmune serum antibodies against various functional domains of various types of toxins prepared in this laboratory, the method is to immunize animals 3-4 times with recombinant Hc, etc.) Afterwards, collect the immune serum antibody) as the primary antibody, and use horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG (Sigma Company) as the secondary antibody, and perform Western blot analysis on each purified recombinant protein (B and B in Fig. 1 E), the results show that the prepared target protein specifically binds to anti-H-type botulinum toxin antibodies such as anti-HHc (B in Figure 1) or HL-HN serum antibody (E in Figure 1), and the positive band size and electrophoretic position It is consistent with the theoretical size of each recombinant protein, indicating that the purified recombinant protein is the target protein. In addition, the present invention has also identified its antigenic cross-reactivity with other serotypes (C and D in Fig. 1), and the results show that anti-A botulinum toxin Hc serum antibodies have obvious cross-reactions with H-type botulinum toxin (Fig. 1, C), does not bind to F-type botulinum toxin Hc, and anti-F-type botulinum toxin L-HN serum antibodies have no cross-reaction with H-type botulinum toxin (Figure 1, D).

In conclusion, the gene sequence of each functional domain of H-type botulinum toxin was cloned by genetic engineering technology, and connected to the prokaryotic expression vector pTIG-Trx to obtain a recombinant expression plasmid. The high-efficiency soluble expression in Escherichia coli purified the recombinant protein with high purity and good stability through HisTrap ^TM HP protein purification column, which provided a basis for the following comparison of the immune protection efficacy of different protein functional domain molecules.

Example 2. Expression, purification and identification of various dual receptor binding region Hc-RBD fusion molecular recombinant proteins in Escherichia coli

1. Gene design and synthesis of various double receptor binding region Hc-RBD fusion molecular recombinant proteins

According to codon degeneracy, artificially optimized and synthesized genes encoding receptor binding domains Hc-RBD (AHc, BHc, EHc and THc) of various types of botulinum toxin and tetanus toxin (see table for details of receptor binding domain Hc and sequence information 2), and first directly cloned in a cloning vector such as pMD18-T vector for future use. When cloning these artificially synthesized genes, in order to facilitate the following connection operations, EcoR I and BamH I restriction sites were introduced at the 5' end of one Hc gene, and BamH I restriction sites were introduced at the 5' end of the other Hc gene. BamH I and 3' end introduced Xho I restriction restriction site.

The above gene design adopts codons commonly used in Escherichia coli, and ensures that the encoded amino acid residue sequence remains unchanged. According to the full-length gene sequence and amino acid residue sequence of each type of botulinum toxin (such as type A botulinum toxin, strain 62A, a full length of 1296 amino acids; type B botulinum toxin, strain Okra, a full length of 1291 amino acids; type E botulinum toxin Toxin, strain Beluga or NCTC11219, full-length 1252 amino acids; tetanus toxin, strain CMCC64008, full-length 1315 amino acids) optimize all types of biotoxin receptor binding region Hc gene, more conducive to the biosynthesis of biotoxin gene protein with expression.

Table 2. Basic information of Hc in the receptor binding region of various types of botulinum toxin and tetanus toxin

2. Construction and identification of recombinant prokaryotic expression vectors

First, in order to facilitate the cloning of multiple target genes into the prokaryotic expression vector pTIG-Trx (see patent: ZL200710089588.2), the present invention first clones BamH I and a connecting peptide Linker-(G4S) ₃ into the EcoR I of pTIG-Trx between Xho I and Xho I, so that the whole vector has three cloning restriction sites (EcoR I, BamH I and Xho I; there is this connecting peptide Linker-(G4S) ₃ in front of BamH I), which can be easily cloned into two different gene, and the expression vector was named pTIG-Trx-M. In addition, in order to enrich the method for purifying the target protein, the present invention also replaces the his tag of the expression vector pTIG-Trx-M with a Twin Strep tag through two restriction sites of Xho I and Sac I (gene sequence TGGAGCCCCCCCAGTTCGAGAAGGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCAGCGCCTGGAGCACCCCCAGTTCGAGAAG and encoding amino acid WSHPQFEK -(GGGS)2-GGSAWSHPQFEK), the expression vector is named pTIG-Trx-M-TS, so the protein of interest can be purified through the Strep-tag label.

Use "EcoR I and BamH I" or "BamH I and Xho I" to double-enzyme-digest each plasmid containing the target gene sequence obtained in the above step 1, respectively recover the corresponding target gene fragments with a DNA recovery kit, and double-enzyme with the same enzyme. Ligate the enzyme-cut prokaryotic expression vector pTIG-Trx-M or pTIG-Trx-M-TS, transform the ligated product into E. coli (E.coli) DH5α competent cells, screen positive clones, extract plasmids, sequence, obtain sequence and insert The recombinant prokaryotic expression vectors with correct positions are named pTIG-Trx-AHc-THc, pTIG-Trx-THc-AHc, pTIG-Trx-AHc-EHc, pTIG-Trx-EHc-AHc, pTIG according to the different insert fragments - Trx-AHc-BHc and pTIG-Trx-BHc-HHc.

3. Expression of recombinant double Hc fusion protein molecules in Escherichia coli and purification and identification of expression products

1. Expression of each recombinant double Hc fusion protein molecule in Escherichia coli and SDS-PAGE detection of the expression product

Transform Escherichia coli BL21 (DE3) competent cells (TIANGEN company) with the recombinant prokaryotic expression vector constructed in step 2, screen positive recombinants, use the recombinant bacteria transformed with pTIG-Trx empty vector as a negative control, and then press 1:100 Ratio: Inoculate the positive recombinant bacteria into 500mL LB liquid medium containing 100mg/mL ampicillin, carry out mass culture at 37°C and _250rpm , and add chemical Inducer IPTG to a final concentration of 0.4mmol/L, 220r/min shaking culture at 30°C for 4-5 hours, or 220r/min shaking culture at 18°C overnight. After the culture, the bacteria were collected by centrifugation, resuspended in 20mM sodium phosphate buffer (pH 8.0), the cells were broken by ultrasonic, and the supernatant was collected by centrifugation for 12% SDS-PAGE detection. The results showed that all the induced recombinant proteins were It has been expressed and can exist in a soluble form, but the target protein band does not appear in the uninduced strain and the induced empty vector control, indicating that the expressed protein may be the target protein, that is, each recombinant antigen.

2. Purification and identification of expression products

The C-terminus of each recombinant toxin protein molecule expressed in step 1 contains six histidine or streptavidin TS tags, so Ni-NTA affinity chromatography column (Pharmacia company) or streptavidin affinity chromatography Column (Dakowei Biotechnology Co., Ltd.) and purify the soluble expression product according to the instructions to obtain the eluted purified protein, and then perform 12% SDS-PAGE detection on the purified protein, and the detection result shows that the purified target protein has been obtained. The obtained target protein was stored at -20°C or -80°C for later use. Figure 2 is a schematic diagram of the structure of the multivalent biotoxin antigen vaccine molecule based on the double receptor binding domain RBD assembly designed and biosynthesized by the patent of the present invention. Figure 3 and Figure 4 show the purification of the above target proteins (THc-linker-AHc, AHc-linker-THc, AHc-linker-EHc, EHc-linker-AHc, AHc-linker-BHc and BHc-linker-HHc) SDS-PAGE electrophoresis identification map.

3. Western blot of expression products

Various types of toxin standard substances from horse source (purchased from China Institute for Food and Drug Control) and polyclonal antibodies against receptor binding regions of various types of toxins (hyperimmune serum antibodies prepared in this experiment) were used as primary antibodies, and horseradish peroxide Enzyme (HRP) labeled rabbit anti-horse IgG (Sigma Company) was used as the secondary antibody, and Western blot was performed on the purified double Hc fusion protein molecules. The results showed that the expressed target protein specifically combined with the antibodies in various types of toxin antitoxin standards. It shows that they all contain the corresponding functional domains of each type of toxin Hc, which is consistent with the theoretical design. B and C in Figure 3 show the molecular identification results of the double Hc fusion protein of tetanus toxin and botulinum toxin type A. The size and position of the positive band are consistent with the electrophoresis position, which is equivalent to the theoretical size of the recombinant protein molecule, indicating that the purified recombinant protein The protein is the target protein and contains two toxin receptor binding domains Hc functional domains.

In conclusion, the gene sequences of the Hc functional domains of various types of toxin receptor binding regions were cloned by genetic engineering technology, and connected to the prokaryotic expression vector pTIG-Trx series to obtain recombinant expression plasmids. Through the optimization of induced expression conditions, the High-efficiency soluble expression and biosynthesis of recombinant plasmids in Escherichia coli, purified recombinant biotoxin protein molecules with high purity and good stability through HisTrapTM HP protein purification column or streptavidin affinity chromatography column (Figure 4), for The following provides a basis for exploring the immune protection efficacy of different biotoxin proteins as subunit vaccines.

Table 3 shows the structural and characteristic information of each dual receptor binding domain Hc fusion molecule finally obtained through this embodiment.

Table 3. Information on the structure and characteristics of Hc fusion molecules in each double receptor binding domain

Note: Each double receptor binding region Hc fusion molecule is composed of two functional domains of the receptor binding region Hc from two different biotoxin sources, and they are connected by a GS linker. The peptide gene sequence is GGCGGTGGCGGTAGTGGCGGTGGCGGTAGCGGCGGTGGCGGTAGTGGATCC; the amino acid sequence is GGGGSGGGGSGGGGSGS.

Example 3. Antibody level detection and protective effect on H-type botulinum toxin after immunizing mice with epitope antigen protein of each functional domain of H-type botulinum toxin

Since H-type botulinum toxin is a new type of serotype toxin, it can also cause human poisoning, and currently there is no specific antibody and vaccine to protect against it. Therefore, the present invention explores the immune efficacy of the epitope antigens of each functional domain of H-type botulinum toxin, as well as its immune cross-reaction characteristics with other serotype botulinum toxin protective antigens.

Each of the recombinant protein antigens prepared in Example 1 above was immunized into mice respectively, and their immunogenicity and protective properties were tested. The specific method is as follows: Balb/c mice (6-8 weeks, female, SPF grade, purchased from the Experimental Animal Center of the Military Medical Research Institute) were randomly divided into groups, 10 in each group, and the immunization dose was 1 μg or 10 μg per mouse. Antigen protein, and negative control group immunization does not contain the PBS of recombinant protein, is all mixed with the aluminum adjuvant (Alhydrogel ^TM 2.0%, the product of Bangtai (BrenntagBiosector) company) that final concentration is 1mg/ml, boosts immunization (antigen protein) at intervals of 3 weeks and the amount of aluminum adjuvant as above), after two or three immunizations, the mice were blood-collected, and then various types of botulinum toxin (BoNT/A/F/H, wherein the present embodiment 3-4 mainly uses H-type botulinum toxin to carry out Protective test, embodiment 5 has used three kinds of botulinum toxins of A, F and H type to carry out protective test) challenge the protective effect of toxin evaluation protein antigen (observe a week, statistical result), and detect mouse serum antibody level with ELISA method And use the classic in vivo neutralization experiment to determine the neutralization titer, the method is as follows.

ELISA method (enzyme-linked immunosorbent plate was used to coat each recombinant antigen at a concentration of 2 μg/mL, the isolated immunized mouse serum was used as the primary antibody, and HRP-labeled goat anti-mouse IgG (Santa Cruz Biotechnology, Inc.) was used as the secondary antibody ) to measure serum antibody levels. The mouse serum of the immunized group diluted with PBS aluminum adjuvant was used as a negative control (ie N), and the OD ₄₉₂ value (ie P) of the immunized group reached more than 0.5, and P/N ≥ 2.1 was positive. Antibody levels in each group were expressed as mean ± standard error.

The in vivo neutralizing activity and neutralizing antibody titer of immunized mouse serum antibodies were determined by classical in vivo neutralization experiments. Serum antibody titers were reported in International Units per milliliter (IU/ml), and one IU was defined as neutralizing 10 ⁴ LD ₅₀ A/F/H-type botulinum toxin neutralizing antibodies (Note: refer to this protocol when determining botulinum toxin neutralizing antibodies, this time mainly use H-type botulinum toxin, other types of neutralizing antibodies only replace For different types of botulinum toxin, refer to this method for determination). Design the following protocol to determine the neutralization titer of mouse serum (refer to the Chinese Pharmacopoeia 2020 edition).

(1) The toxin is diluted with the sample to be tested. Dilute the toxin to 100LD ₅₀ /ml. Then the mouse serum was diluted to a multiple corresponding to the corresponding toxin according to the expected titer, for example, the estimated 2IU/ml serum was diluted 200 times to 0.1IU/ml. At the same time, dilute the antitoxin standard to the corresponding concentration.

(2) The toxin is combined with the sample to be tested in vitro. 1ml of the diluted toxin was mixed with different volumes of diluted serum or antitoxin standard (purchased from China Institute for Food and Drug Control), and 5 gradients were set up for each group. Make up to a final volume of 2.5ml with diluent, mix the toxin and antibody evenly, and incubate in a 37°C incubator for 15 minutes to fully react the toxin and antibody.

(3) Administration and observation of animals. SPF grade female KM mice of 15-18 g were randomly divided into four groups. The incubated biotoxin combined with antitoxin standard or test sample was intraperitoneally injected into mice at a dose of 500 μl/mouse, and 4 mice were administered for each dilution of each sample. The morbidity and mortality of animals were observed every day for 7 consecutive days. The 50% protection endpoint of the animals in the experimental group was compared with the 50% death endpoint of the biotoxin standard animal group, and the titer of the serum was calculated (refer to the 2020 edition of the Chinese Pharmacopoeia).

The results shown in A in Table 4 and Figure 5 show that the recombinant antigens of HL, HHN, HL-HN and HHc all produced their own antibody responses, and each immunization group had different doses and immunization times. positive correlation. However, the protective effects against the toxin and the levels of neutralizing antibodies were different. Different doses and immunization times of HL-HN and HHc immunized groups produced strong protective effects, while HHN, a weaker protein antigen, produced partial protective effects. , HL has no protective effect.

Table 4. The results of the protection level of each functional domain epitope antigen protein of H-type botulinum toxin after immunizing mice

Note: 2× indicates the second immunization group, 3× indicates the third immunization group; 3 weeks after the last immunization, different doses of H-type botulinum toxin were used to challenge the protective experiment; the survival number/total number indicates the protection level, and each group 10 test animals. Serum neutralizing antibodies were determined using classical in vivo neutralization assays. In terms of protection level, HL group and HN group had extremely significant statistical difference compared with Hc group and HL-HN group ( ^*** p=0.00001<0.001). ND indicates that there is no protective test for this dose group.

Example 4, Botulinum Toxin Type H Functional Domain Hc and L-HN Protein Antigen Dose-Dependent Immunoprotective Results

In order to further evaluate the protective efficacy of THc and TL-HN at low doses, the present invention conducted a protective test study on these two protein antigens after immunization once or twice with different doses for three weeks. Type H botulinum toxin recombinant Hc and L-HN antigen dose-dependent immune protection scheme is as follows: the doses of recombinant Hc and L-HN antigen and Hc+L-HN combination group were 4000, 1000, 250, 62.5, 15.6 and 3.9ng/only, immunization 1 time or 2 times, others are the same as the scheme in the above embodiment 3. The 1st immunization group was challenged with 10 ² LD ₅₀ toxin three weeks after the first immunization, and the 2nd immunization group was challenged with 10 ³ LD ₅₀ toxin three weeks after the second immunization, observed for one week, and counted the results of survival. Use the probability analysis method (SPSS17.0-Probit) to calculate the half effective dose ED ₅₀ value of each group, and use this value to represent the efficacy of the vaccine.

The results shown in Table 5 show that after the three immunization groups of HHc, HL-HN and HHc+HL-HN were immunized with different doses, they all produced a strong protective effect, and HHc, HL-HN and HHc+HL after one immunization The half effective dose ED ₅₀ of the -HN group was 96.419ng, 673.105ng and 32.144ng respectively; the half effective dose ED ₅₀ of the HHc, HL-HN and HHc+HL-HN groups after two immunizations were 2.193ng, 8.262ng respectively and 4.478ng.

The HHc antigen group produced a stronger protective effect than the HL-HN antigen group, indicating that it is the strongest subunit vaccine antigen. The HHc+HL-HN antigen group had the highest protective effect after 1 immunization, indicating that the combination group had a synergistic effect compared with the HHc and HL-HN alone groups, reflecting more neutralizing antibody epitopes induced a stronger immune effect ; while the HHc antigen group had the strongest protective effect after two immunizations. The above results further show that HHc is far superior to HL-HN as an immune antigen, especially under the condition of low dose and few times of immunization, its effect is more obvious. Therefore, the results of the present invention validate and support HHc as its protective antigen.

Table 5. The results of protection level after immunizing mice with different doses of botulinum toxin recombinant HHc and HL-HN antigens

Note: The HHc+HL-HN group was combined immunization with two antigen groups; the 1-time immunization group received 10 ² LD ₅₀ toxin three weeks after the first immunization, and the 2-time immunization group received 10 ³ Attack with LD ₅₀ toxin, observe for a week, and count the survival results.

Example 5, Protective effect of H-type botulinum toxin functional domain epitope antigen protein on mice after immunization with A, F and H-type botulinum toxin

In view of the fact that HL-HN and HHc functional domains are important protective antigens, and they have sequence homology with A and F-type botulinum toxin-related functional domains, the present invention further explores their relationship with A and F-type botulinum toxins. Cross-protective effects of toxic toxins. The experimental scheme is the same as in Example 3, that is, the immunization doses of HL-HN and HHc are 1 μg or 10 μg of antigenic protein per mouse respectively, and booster immunization at intervals of 3 weeks (the amount of antigenic protein and aluminum adjuvant is the same as above), and after two or three immunizations, the Mice were bled and tested for protection.

The results shown in B in Figure 5 indicate that HL-HN and HHc immune serum antibodies have cross-antibody reactions with FL-HN and AHc, which is related to their high sequence homology with FL-HN and AHc. However, HL-HN and HHc functional domain antigens were immunized with 1 μg and 10 μg doses for 2 and 3 times for protective test evaluation. The results showed that HL-HN recombinant antigens could not resist the attack of 100LD ₅₀ F type botulinum toxin, without any Protective effect; while the HHc antigen can completely resist the attack of 1000LD ₅₀ type A botulinum toxin, and the immune serum also contains high titers of neutralizing antibodies. The neutralizing antibodies against type H botulinum toxin were 2.0 and 10.0 IU/ml after the HHc functional domain was immunized twice with doses of 1 μg and 10 μg, respectively, and the neutralizing antibodies against type A botulinum toxin were 0.5 and 4.0 IU/ml, respectively , indicating that there are cross-protection neutralizing antibodies between them. Therefore, the above results suggest that the protective effect is directly related to neutralizing antibodies.

In addition, the present invention continues to carry out research on the dose-dependent cross-immune protection of Hc-type botulinum toxin recombinant Hc and A-type botulinum toxin recombinant Hc. The scheme is as follows: the doses of recombinant HHc and AHc antigens are 4000, 1000, 250, 62.5, 15.6 and 3.9 ng/monkey in sequence, immunized twice, and the others are the same as the scheme in Example 3 above. Each immunization group was challenged with BoNT/A and BoNT/H after immunization once and twice three weeks later, observed for one week, and counted the number of survivors. The results are shown in Table 6. The results of protective experiments showed that HHc and AHc not only produced a protective response against their own toxin, but also produced a strong cross-protective response with botulinum toxin type A or botulinum toxin H, and this cross-protective effect also showed Antigen dose dependence. The half effective dose ED ₅₀ of the cross-protective effect in the HHc and AHc groups after one immunization was 40.479ng and 1122.781ng respectively; the half effective dose ED ₅₀ of the cross-protective effect in the HHc and AHc groups after two immunizations were 32.144ng and 55.816ng respectively

Therefore, the results of the present invention show that Hc, including HHc and AHc, is the dominant protective antigen molecule, and has obvious cross-protection with them, and can be used to prepare vaccines and protect against each other's toxins. In our laboratory, the anti-AHc botulinum anti-toxin antibody we prepared can neutralize H-type botulinum toxin, and it needs 4 times the anti-A-type botulinum toxin neutralizing antibody to completely neutralize the same amount of H-type botulinum toxin toxin.

At the same time, the study found that the L-HN of BoNT/H is unique, it has no cross-reaction with the F-type botulinum toxin L-HN protective antigen, and cannot protect F-type botulinum toxin, so it is in the L-HN functional domain It is obviously different from the existing botulinum toxin. The existing traditional antitoxin cannot neutralize the toxin and needs 500 times more neutralizing antibodies to neutralize it. Therefore, this antibody drug cannot be used to specifically treat it .

Table 6. Cross-protection results after Hc antigen protein immunization of H-type and A-type botulinum toxin receptor binding regions

Note: In the HHc immunization group, the protective test was carried out with 10 ² LD ₅₀ BoNT/A three weeks after the first immunization in the 1st immunization group; 10 ³ LD 50 BoNT/A was used in the 2nd immunization group ₅₀ BoNT/A for protective test; in the AHc immunization group, the protective test was carried out with 10 ² LD ₅₀ BoNT/H three weeks after the first immunization in the AHc immunization group, and the second immunization three weeks after the second immunization group Then use 10 ³ LD ₅₀ BoNT/H to conduct a protective test; observe for a week, and count the results of survival.

In summary, the present invention utilizes genetic engineering technology to prepare protein molecules of each functional domain of H-type botulinum toxin, and explores the immune protection efficacy of each recombinant protein antigen subunit vaccine. The results show that H-type botulinum toxin each Among functional domain protein antigens, Hc and L-HN antigens have good immunogenicity and immune protection, HN antigen has general immune protection, and L has no protective effect. The results of further research and analysis showed that HL-HN had no cross-protective effect with F-type botulinum toxin, while HHc had a strong cross-protective effect with A-type botulinum toxin. Compared with L-HN antigen, Hc antigen had a moderate protective effect and And antibody production has obvious advantages and better potency.

Therefore, the efficiency of the recombinant Hc protein antigen prepared by the present invention as H-type botulinum toxin subunit vaccine is better than that of L-HN antigen, and has better immune protection effect and application prospect. The Hc is not only an effective protective antigen, but also has a cross-protective effect with the type A botulinum toxin, and can be used to construct a double Hc fusion molecule as a multivalent vaccine.

Example 6, Tetanus toxin and botulinum toxin type A double Hc fusion molecular protein as the immune effect of multivalent subunit vaccine

The recombinant protein antigens prepared in the above Example 2 were immunized with mice respectively, and their immunogenicity and protective properties were tested. The specific method is: divide Balb/c mice (6-8 weeks, female, SPF grade) into random groups, 10 mice in each group, and the immunization dose is 1 μg or 4 μg antigen protein per mouse, while the negative control group is immunized without The PBS of the recombinant protein was mixed with aluminum adjuvant (Alhydrogel ^TM 2.0%, product of Brenntag Biosector) with a final concentration of 1 mg/ml, and boosted immunization at intervals of 3 weeks (the amount of antigenic protein and aluminum adjuvant was the same as above), one Or blood was collected from mice after two immunizations, and then challenged with different biological toxins to evaluate the protective effect of protein antigens.

The results shown in Table 7 and Figure 6 indicated that both AHc-THc and THc-AHc recombinant protein antigens produced antibody responses against themselves, as well as immune protection against AHc and THc. Different doses and times of immunization in each immunization group produced protective responses, and also showed a dose-positive correlation. After one immunization, the protective effect against tetanus toxin was slightly weaker, but after two immunizations, both AHc-THc and THc-AHc groups produced complete protection against tetanus toxin, and had the same protective effect as the control group AHc+THc, It shows that there is no influence between AHc and THc in the double Hc fusion molecule, and both combinations can exert immune effects. In particular, the double Hc fusion molecule as a vaccine produced strong antibody responses against AHc (B in Figure 6) and THc (C in Figure 6), further reflecting that the two antigenic components in the fusion molecule played their respective roles .

In order to further evaluate the protective efficacy of AHc-THc and THc-AHc at low doses, the present invention carried out protective experimental research after immunizing these two protein antigens once or twice with different doses. The immunoprotective scheme is as follows: the dosage of each recombinant protein antigen is 1000, 250, 62.5, 15.6 and 3.9ng/monkey in turn, immunized once or twice, and other schemes are the same as above. After three weeks of immunization, each immunization group was challenged with different biological toxins, observed for one week, and counted the survival results. Use the probability analysis method (SPSS17.0-Probit) to calculate the half effective dose ED ₅₀ value of each group, and use this value to represent the efficacy of the vaccine.

The results shown in Table 8 to Table 10 show that after THc-AHc and AHc-THc and the control AHc+THc combination group were immunized with different doses, they all produced strong protective effects against the two toxins, and the three groups had a strong protective effect in terms of protective efficacy. No difference. For tetanus toxin, the half effective doses ED ₅₀ of the three vaccines after one immunization are 8.17ng, 31.45ng and 21.104ng respectively; the half effective doses ED ₅₀ of the three vaccines after two immunizations are 8.17ng, 21.104ng respectively and 31.225ng. For botulinum toxin type A, the half effective doses ED ₅₀ of the three vaccines after one immunization were 223.721ng, 31.45ng and 31.45ng respectively; the half effective doses ED ₅₀ of the three vaccines after two immunizations were 8.17ng, 8.17ng and 8.17ng.

In addition, AHc-THc and THc-AHc molecular vaccines can also completely protect H-type botulinum toxin after two immunizations, indicating that this RBD-based double fusion molecule can also produce cross-protection against H-type botulinum toxin. Therefore, single-molecule AHc-THc and THc-AHc can be used as multivalent vaccine molecules to replace combined group antigens, which can protect three toxin pathogens, and are vaccine varieties with good application prospects.

Table 7. Immunoprotective effects of tetanus toxin and botulinum toxin type A double Hc fusion molecular vaccine against the two toxins

Note: 3 weeks after the last immunization, different doses of botulinum toxin type A and tetanus toxin were challenged for protective experiments; serum neutralizing antibodies were determined by classical in vivo neutralization experiments. Survival number/total number indicates protection level, wherein each group protects 10 animals.

Table 8. Results of protection levels against two toxins after immunizing mice with different doses of double Hc fusion molecule THc-linker-AHc

NOTE: 3 weeks after the last immunization, challenge with different doses of botulinum toxin type A versus tetanus toxin for protective experiments. Survival number/total number indicates protection level, wherein each group protects 10 animals.

Table 9. The results of protection against two toxins after immunizing mice with different doses of the double Hc fusion molecule AHc-linker-THc

NOTE: 3 weeks after the last immunization, challenge with different doses of botulinum toxin type A versus tetanus toxin for protective experiments. Survival number/total number indicates protection level, wherein each group protects 10 animals.

Table 10. The results of the protection levels against the two toxins after mice were immunized with different doses of AHc+THc combined vaccine

NOTE: 3 weeks after the last immunization, challenge with different doses of botulinum toxin type A versus tetanus toxin for protective experiments. Survival number/total number indicates protection level, wherein each group protects 10 animals.

Example 7. Research on the immune effect of other types of botulinum toxin double Hc fusion molecule protein as multivalent subunit vaccine

Based on the multivalent biotoxin protein biosynthesis technology of double receptor binding domain RBD assembly, the present invention also carried out the combination research of other types of receptor binding domain RBD. Firstly, the immune effect of type A and E botulinum toxin double Hc fusion molecule protein vaccine (AHc-EHc and EHc-AHc, A in Figure 4) was explored. The immunization and program evaluation are the same as in Example 6, with 5 mice in each group, and the immunization dose is 1 μg or 10 μg of antigenic protein per mouse, and the immunization is boosted at intervals of 3 weeks. After one or two immunizations, blood is collected from the mice, and then different biological toxins are used. The protective effects of protein antigens were evaluated by challenge.

The results in Table 11 show that after AHc-EHc and EHc-AHc are used as subunit vaccines to immunize animals, they induce a strong protective effect against botulinum toxin type A, but the protective effect against botulinum toxin type E is weak and requires immunization 3 times can provide a certain protection. However, when the AHc+EHc combination group was immunized, the protective efficacy against type A and E botulinum toxins was strong. These results suggested that the EHc protective antigen in the double Hc fusion molecule of A and E botulinum toxin was weakened, and this effect It may be related to the fact that botulinum toxin Hc is a weak protective antigen in the functional domain of botulinum toxin E, and L-HN is a strong protective antigen (see patent ZL 201911292144.8 for details). Therefore, when studying botulinum toxin type E and other types of toxins to assemble multivalent vaccines, it is necessary to select EL-HN as a protective antigen. This theory and technology have been confirmed by our team.

Therefore, when combining double Hc fusion molecules targeting various types of toxins, it is necessary to consider the strength of their RBD as a protective antigen to support strong fusion assembly. In order to verify this technology, the present invention carried out more confirmatory studies, such as A or H and B type botulinum toxin double Hc fusion molecule protein as a multivalent subunit vaccine biosynthesis (B in Figure 4) and its immunization effect research. The immunization dose was 1 μg or 10 μg antigenic protein per mouse, and the immunization was boosted at intervals of 3 weeks. Three weeks after the two immunizations, different biological toxins were challenged to evaluate the protective effect of the protein antigen. The research results show that other types of combinations such as AHc-BHc and BHc-HHc can also produce strong protective effects against botulinum toxin types A, B, and H, and the protective efficacy against the three toxins is equivalent, and there is no difference in strength or weakness. points (Table 12). Previously, this study confirmed that A or H type Hc has strong cross-protection due to high homology, and there is no protective difference between them. Both A type Hc antigen or H type Hc antigen can completely protect A and H types of botulinum toxin toxin. The results of the double Hc fusion molecular protein multivalent subunit vaccine once again proved that A or H type Hc in the fusion molecule has strong cross-protection, and also confirmed that they can be used as a trivalent vaccine. The above results showed that the feasibility of strong combination was further verified in the combination of A or H and B type double Hc. Therefore, the effectiveness of more combination candidate vaccine molecules can be further verified according to this principle.

In a word, the double Hc fusion antigen molecular vaccine of the present invention can produce protective effects against two or three different serotype toxin pathogens, and can be used as a broad-spectrum multivalent vaccine for the biological defense of toxins.

Table 11. Immunoprotective effect of the double Hc fusion molecule protein vaccine of type A and E botulinum toxin against two toxins

NOTE: 3 weeks after the last immunization, challenge with different doses of botulinum toxin types A and E for protective experiments. Survival/total represents the level of protection, with 5 test animals per group. ND indicates that there is no protective test for this dose group.

Table 12. Immunoprotective effects of botulinum toxin double Hc fusion molecule vaccine against three toxins

NOTE: 3 weeks after the two immunizations, challenge with 10 doses of each of the ^three botulinum toxins for protective experiments. Survival number/total number indicates protection level, wherein each group protects 10 animals.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

Claims (10)

1. A multivalent biotoxin molecular antigen vaccine, the active ingredient of which is a fusion protein formed by linking the Hc antigen of biotoxin 1 and the Hc antigen of biotoxin 2 through a connecting peptide; The biotoxin 1 and the biotoxin 2 are two different biotoxins; Said biological toxin is selected from the following: tetanus toxin or other types of botulinum toxin other than type E. 2. The multivalent biotoxin molecular antigen vaccine according to claim 1, characterized in that: the non-E-type other types of botulinum toxin are A-type botulinum toxin or B-type botulinum toxin or H-type botulinum toxin . 3. The multivalent biotoxin molecular antigen vaccine according to claim 1 or 2, wherein the active ingredient of the multivalent biotoxin molecular antigen vaccine is any of the following: (A1) A fusion protein formed by linking the Hc antigen of botulinum toxin type A and the Hc antigen of tetanus toxin through a linking peptide in the order from amino acid to carboxyl terminal, and the fusion protein is named AHc-THc; (A2) A fusion protein formed by linking the Hc antigen of tetanus toxin and the Hc antigen of botulinum toxin through a linking peptide in the order from amino acid to carboxyl terminal, and the fusion protein is named THc-AHc; (A3) A fusion protein formed by linking the Hc antigen of botulinum toxin type A and the Hc antigen of botulinum toxin type B through a linking peptide in the order from the amino acid to the carboxyl terminal, and the fusion protein is named AHc-BHc; (A4) A fusion protein formed by linking the Hc antigen of botulinum toxin type B and the Hc antigen of botulinum toxin type H via a linker peptide in the order from the amino acid to the carboxyl terminal. The fusion protein is named BHc-HHc. 4. The multivalent biotoxin molecular antigen vaccine according to any one of claims 1-3, characterized in that: the connecting peptide is (G4S) ₃ . 5. The multivalent biotoxin molecular antigen vaccine according to any one of claims 1-4, characterized in that: the amino acid sequence of the Hc antigen of the type A botulinum toxin is as shown in SEQ ID No.10; and/ or The amino acid sequence of the Hc antigen of the botulinum toxin type B is shown in SEQ ID No.12; and/or The amino acid sequence of the Hc antigen of the H-type botulinum toxin is shown in SEQ ID No.8; and/or The amino acid sequence of the Hc antigen of the tetanus toxin is shown in SEQ ID No.16; and / or Further, the amino acid sequence of the AHc-THc is shown in SEQ ID No.18; and/or Further, the amino acid sequence of the THc-AHc is shown in SEQ ID No.20; and/or Further, the amino acid sequence of the AHc-BHc is shown in SEQ ID No.26; and/or Further, the amino acid sequence of the BHc-HHc is shown in SEQ ID No.28. 6. The method for preparing the polyvalent biotoxin molecular antigen vaccine described in any one of claims 1-5, comprising the steps of: introducing the nucleic acid molecule encoded as the fusion protein into E. coli recipient cells to obtain recombinant E. coli; culturing The recombinant Escherichia coli is obtained as the fusion protein; then the multivalent biotoxin molecular antigen vaccine is prepared by using the fusion protein as an active ingredient. 7. The method according to claim 6, characterized in that: the nucleic acid molecule sequence encoding the Hc antigen of the type A botulinum toxin is as shown in SEQ ID No.9; and/or The nucleic acid molecular sequence encoding the Hc antigen of botulinum toxin type B is shown in SEQ ID No.11; and/or The nucleic acid molecule sequence encoding the Hc antigen of the H-type botulinum toxin is shown in SEQ ID No.7; and/or The nucleic acid molecular sequence encoding the Hc antigen of the tetanus toxin is shown in SEQ ID No.15. 8. The method according to claim 6 or 7, characterized in that: the nucleic acid molecule sequence encoding the AHc-THc is as shown in SEQ ID No.17; and/or The nucleic acid molecule sequence encoding the THc-AHc is shown in SEQ ID No.19; and/or The nucleic acid molecule sequence encoding the AHc-BHc is shown in SEQ ID No.25; and/or The nucleic acid molecule sequence encoding the BHc-HHc is shown in SEQ ID No.27. 9. Any of the following substances: (B1) protein, which is the fusion protein described in any one of claims 1-5; (B2) a nucleic acid molecule, which is the nucleic acid molecule described in any one of claims 6-8; (B3) recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the nucleic acid molecules described in (B2); (B4) A product for the prevention and/or treatment of poisoning symptoms caused by botulinum toxin and/or tetanus toxin, containing the protein described in (B1) or the nucleic acid molecule described in (B2) or the nucleic acid molecule described in (B3) The above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria. 10. The protein described in (B1) or the nucleic acid molecule described in (B2) or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria described in (B3) in claim 9 is used for prevention and/or treatment Application in products for intoxication conditions caused by botulinum toxin and/or tetanus toxin.

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