CN107177539A - A kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine - Google Patents

Abstract

The invention provides a method for constructing a spore-type probiotic mucosal vaccine and an immune evaluation system, and provides a double-transposition expression vector capable of expressing exogenous antigens of pathogenic organisms and a host bacterium. The invention also provides a fusion The probiotic vaccine carrier expressing cytokine IL-2, the present invention also provides a kind of Schistosoma japonicum vaccine and bacillus tetani bivalent vaccine, and a kind of Schistosoma japonicum monovalent vaccine, and described probiotic vaccine uses Bacillus subtilis as oral Or mucosal delivery vehicle for nasal drop route. For the first time, the present invention organically combines exogenous genes such as Schistosoma japonicum Sj26GST and tetanus TTFC, cytokine IL-2 and Bacillus subtilis mucosal immune vaccine carrier to complement each other. Sj26GST and TTFC were selected as model antigens, spore vectors were used to deliver model antigens, and recombinant IL-2 displayed through spores and spores was used as an adjuvant to adjust the type of immunity and enhance the level of immune response.

Description

A Bivalent Oral or Nasal Vaccine against Schistosoma japonicum and Tetanus

technical field

The invention relates to a vaccine, in particular to a bivalent vaccine with IL-2 as an adjuvant and spore-type probiotics as a mucosal delivery carrier.

Background technique

Using a new generation of nano-scale biological carrier, that is, nano-scale spores of Bacillus subtilis as a mucosal delivery vehicle to develop oral or nasal vaccines, it is estimated that industrial production within one month can meet the needs of high-risk populations for immunity. The spore vaccine produced by fermentation technology has greatly shortened the culture time compared with traditional chicken embryo vaccine, and the estimated unit cost per dose is only 1/10 of the production cost of traditional methods. Therefore, new vaccines with short research and development cycle, fast production, low cost, strong anti-interference ability to the external environment, easy storage and transportation, long-lasting cross-protection effect, and adjuvant effect have become urgent for the prevention and control of infectious diseases worldwide. need. Vaccines constructed using Bacillus subtilis as a mucosal vaccine delivery vehicle can meet these needs.

The present invention utilizes endophytic spores of Bacillus subtilis to construct a multivalent antigen fusion expression system, selects Schistosoma japonicum 26kDa GST (Sj26GST) protein as a model antigen and tetanus toxin C fragment (TTFC) as an immune adjuvant molecule, and displays it on the outer surface of the spores (CotC-Sj26GST-TTFC expressed in fusion with spore CotC coat protein); and expressed in a way combined with double transfection construction for displaying IL-2 on the outer surface of spore (expressed in fusion with spore CotB-IL-2 coat protein). Fluorescence immunology experiments and laser confocal microscopy detected that foreign proteins were successfully expressed on the outer surface of spores, which also proved that the macromolecular structure of multivalent recombinant antigens did not affect the assembly of spore outer layer proteins and spore formation, and proved that IL- 2 can improve the level of cellular immunity of the vaccine.

Oral or intranasal immunization with synthetic or recombinantly expressed spores can elicit a broad immune response, but the level of CD8+ T cell immunity induced is relatively low. Therefore, the present invention utilizes interleukin-2 (IL-2) to regulate the type of immunity and enhance the level of cellular immunity.

Interleukins (interleukins, ILs) ILs are cytokines that mediate the interaction between leukocytes and other cells. Cytokines produced by lymphocytes, monocytes, or other non-monocytes play important regulatory roles in cell-cell interactions, vaccine immune regulation, hematopoiesis, and inflammatory processes. Interleukin-2 (IL-2) is mainly produced by CD4+T cells or CD8+T cells, and exerts its effects in an autocrine and paracrine manner. The main biological functions include: ① activate CD4+ and CD8+ T cells, promote cytokine production; ② stimulate the proliferation and activation of NK cells, induce the production of LAK cells; ③ promote the proliferation of activated B cells and produce antibodies; Phage cells. IL-2 is a human body's own component, which can regulate the body's physiological process and improve immune function. It can play a role at a very low dose, with significant curative effect and small side effects. The recombinant cytokine IL-2 produced by genetic engineering technology is used as a biological response regulator. (biological response modifier, BRM) has also achieved a certain effect in the treatment of infectious diseases. It is a brand-new biological agent and has become a new generation of drugs approved for production.

The invention introduces the biological response regulator IL-2 to exert its immune regulation adjuvant effect, regulate the type of immune response, enhance the level of cellular immunity, so as to improve the immune protection effect of exogenous recombinant antigen vaccine.

The novel nano-scale biological carrier Bacillus subtilis used in the present invention belongs to Gram-positive bacteria, has non-pathogenicity, is widely distributed, usually exists in soil, and is approved by the U.S. Food and Drug Administration (FDA) as "conventional safety level" (generally recognized as safe, GRAS), often used as probiotics in food or medicine. Bacillus subtilis is widely used in the fields of scientific research and industrial and agricultural production. It has a good fermentation foundation and production technology, and it can grow to a high density in a relatively simple medium. Many commercial enzymes are extracellular proteins produced by Bacillus, such as α-amylase, protease, and the insecticidal crystal protein of Bacillus thuringiensis. Bacillus grows rapidly, the culture conditions are simple, and the genetic background is clear. The spore of Bacillus is a dormant body (the spore formation process shown in Figure 1), which can maintain a high degree of stability under external resistance conditions, including thermal stability, and can resist harsh environmental conditions.

These characteristics of spores are attributed to their unique spore capsid structure. The capsid multilayer structure of spores is composed of more than 70 kinds of capsid proteins (Cot proteins) (as shown in Figure 2), which form the inner shell, outer shell and outermost layer of spores. The cortical crust capsid layer. Among them, the three outermost capsid proteins CotB, CotC and CotG are often used for fusion expression of foreign antigen proteins or enzymes, which are displayed on the surface of spores.

Bacillus subtilis has unique immunological characteristics, and is often used as a nano-scale biological carrier for recombinant or non-recombinant mucosal vaccines: ①The diameter of the spore is about 0.6-1.0 μm. It can recognize tract-associated lymphoid tissue (GALT) or nose-associated lymphoid tissue (NALT), play the role of immune adjuvant, activate the relevant lymphoid tissue of intestinal mucosa, enhance the secretion of secretory (SIgA) antibody, improve the ability of immune recognition, and induce T, B lymphocytes and macrophages produce cytokines, which activate the systemic immune system through lymphocyte recycling, thereby enhancing the body's non-specific and specific immune systems. ②Spores have the adjuvant effect of immune regulation, and can induce a balanced Th1/Th2 mixed immune response, which can not only activate specific humoral immune response, but also specific cellular immune response. The spore capsid display system is widely used in the research of recombinant mucosal vaccines expressing foreign antigenic proteins; in addition, due to its bioadhesiveness, it is also used in the research of adsorbed vaccine carriers. At the same time, spores have an immune adjuvant effect and can be used to enhance the immune effect of DNA vaccines, polypeptide vaccines, genetically engineered protein vaccines, and inactivated virus particle vaccines, thereby saving a single immune dose of foreign proteins and improving the immune protection effect. Therefore, Bacillus subtilis is considered to be the most promising mucosal vaccine carrier and is widely used in the development of pathogenic microorganism vaccines.

At present, there is no report on the application of Sj26GST-TTFC/IL-2-Bacillus subtilis recombinant mucosal vaccine. If the cytokine IL-2 is expressed through the fusion of the spore vector CotB capsid protein, it will be displayed on the surface of the spore capsid, and it will be double-expressed on the surface of the spore by fusion with the exogenous bivalent antigen gene. In this way, it can not only exert the immunomodulatory effect of IL-2, enhance Th1 immune response, improve the immune level of specific CD8+ T cells, but also exert the adjuvant effect of spores to enhance the intensity of immune response.

According to vaccine development technology, vaccines can be divided into traditional vaccines and new vaccines. In the research and use of new vaccines, it was found that vaccines prepared only with antigens obtained by gene cloning techniques were not enough to achieve effective immune protection. It is suggested that a truly effective vaccine not only depends on the cloning of an antigen that can induce immune protection, but also on the construction of an excellent carrier system, as well as the research and evaluation of the mechanism and approach of antigen-induced immune protection. In recent years, the study of new vaccine vectors to obtain safe, stable and effective new vaccines has become a focus of attention. The vectors used to prepare new vaccines include recombinant bacteria, recombinant viruses, DNA vectors, RNA vectors, dendritic cells, T cells and polypeptides. The diversification of the new vaccine carrier platform provides a variety of antigen delivery methods, and also enriches the immunization route, so that long-lasting immunity can be produced at the infection site and the ideal type of immune protection can be induced according to the needs. Therefore, the application of the novel antigen presentation platform is expected to induce broad-efficiency and long-lasting immune protection, while shortening the time for vaccine development and production.

Bacillus subtilis has unique advantages as a new type of vaccine carrier, and is considered to be the most promising mucosal vaccine carrier, and has been applied to the development of pathogenic microorganism vaccines. Isticato and Mauriello et al. used tetanus toxin C fragment (TTFC, 51.8kDa) and Escherichia coli enterotoxin B subunit (LTB, 12kDa) genes tetC and eltB to encode Bacillus subtilis capsid protein CotB or CotC gene fusion, utilize CotB or The CotC promoter expresses the fusion protein. Fusion proteins CotB-TTFC, CotC-TTFC and CotC-LTB were successfully expressed stably on the surface of spores. Studies have shown that fusion expression did not affect the protection of antigens, nor did it destroy the characteristics of spores. Oral immunization of mice with recombinant spores can induce systemic and local immune responses against TTFC or LTB. Vaccine evaluation studies showed that after oral immunization of mice with CotB-TTFC spores, serum IgG was significantly higher than that of the control group, and it could resist 20 times the challenge infection of TTFC. Hinc et al. used the fusion of spores expressing IL-2 as an immune adjuvant, which can regulate Th1-based immune responses, express or absorb foreign antigens through recombinant fusion, produce specific cellular immune responses, and have certain immunity to Helicobacter pylori. Protective effect. In recent years, with the understanding of the adhesion of spores, subtilis spore vectors have been explored in the development of influenza and avian influenza vaccines: Song used inactivated subtilis spores as an adjuvant and antigenic protein carrier for mucosal immunization to mix and adsorb with HA of H5N1 virus , showing a significant immune regulation adjuvant effect, can enhance the level of immune response, and regulate Th1-based Th1/Th2 mixed immune response, and has a protective effect against avian influenza virus attack. Guangyu Zhao et al expressed the M2e protein of influenza virus A through the fusion of spore capsid CotB protein, oral immunization can produce strong and durable specific systemic immunity and mucosal immunity, completely resisting the attack of influenza virus H1N1.

The traditional vaccine development technology can no longer meet the needs of today's vaccines. It is necessary to combine a variety of new concepts, new technologies, and high-efficiency vaccine platforms to achieve the goal together. Through the comprehensive research of model antigen, IL-2 recombinant spore and subtilis spore mucosal vaccine platform, the present invention provides technical support and theoretical basis for the research and development of oral or nasal vaccine with lasting immune protection for Schistosoma japonicum vaccine and tetanus vaccine.

The development cycle is short, the production cost is low, easy to store and transport, the immunization method does not require injection, can produce a long-lasting cross-protection effect, activate humoral immunity and cellular immunity at the same time, and a new type of vaccine with its own adjuvant effect has become an urgent need in the world .

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a host bacterium. The present invention also provides a bivalent oral or nasal vaccine for Schistosoma japonicum and tetanus, the bivalent vaccine is spore-type Probiotics are mucosal delivery vehicles.

In order to achieve the above purpose, the technical solution adopted: the invention provides a host bacterium, which is prepared by the following method:

(1) The nucleotide sequence of the CotB gene promoter sequence, the CotB coding sequence, the linker and the nucleotide sequence of IL-2 are sequentially connected to the first expression vector to obtain the first expression vector of the cotB-linker-IL2 fusion protein Recombinant expression vector;

(2) In the step (1), single enzyme digestion outside the insert fragment of the first recombinant expression vector makes the linearization of the first recombinant expression vector, and then transforms into the first Bacillus competent cell, and screens to obtain the expression of CotB-linker - the first positive strain of IL-2 fusion protein;

(3) connecting the CotC gene promoter sequence, the CotC coding sequence and the nucleotide sequence of the above-mentioned Sj26GST-TTFC to the second expression vector in sequence to obtain the second recombinant expression vector expressing the CotC-Sj26GST-TTFC fusion protein;

(4) In the step (3), single enzyme digestion outside the insert fragment of the second recombinant expression vector makes the linearization of the second recombinant expression vector, and then transforms into the second Bacillus competent cell, and screens to obtain the expression of CotC-Sj26GST - the second positive strain of the TTFC fusion protein;

(5) Extract the chromosome of the second positive strain expressing the CotC-Sj26GST-TTFC fusion protein in the step (4), and transform it into the IL-2CotB-linker-IL-2 fusion protein expressed in the step (2). The competent cells prepared from the first positive strain were screened for positive clones expressing CotB-IL2 fusion protein and CotC-Sj26GST-TTFC fusion protein at the same time to obtain the host bacteria.

Preferably, the amino acid sequence of the linker is shown in SEQ ID NO.1.

Preferably, the nucleotide sequence of Sj26GST-TTFC in the step (3) is as shown in SEQ ID NO.6

Preferably, the first expression vector in step (1) is plasmid pDG364, and the second expression vector in step (3) is plasmid pDG1664.

Preferably, the first Bacillus competent cell in the step (2) is a Bacillus subtilis PY79 competent cell, and the second Bacillus competent cell in the step (4) is a Bacillus subtilis PY79 competent cell.

The present invention provides the use of the above-mentioned host bacteria in the preparation of bivalent oral or nasal vaccines against Schistosoma japonicum and tetanus.

The invention provides a bivalent oral or intranasal vaccine of Schistosoma japonicum and tetanus, which contains spores cultured from the above-mentioned host bacteria.

In the present invention, on the established probiotic Bacillus subtilis mucosal vaccine platform, in order to induce the type of immune response dominated by specific CD8+ T cells, the Th1 immune response is enhanced through the regulation of cytokine IL-2, thereby increasing the specificity of CD8+ T cells. + T cell immune response level. Through the fusion expression of the CotB capsid protein of the spore vector, the cytokine IL-2 is displayed on the surface of the spore capsid, so that it can exert an adjuvant effect, regulate the type of immune response, enhance cellular immunity, prevent and completely eliminate pathogenic microorganisms that infect the body; The spore capsid protein CotB is fused to express IL-2, and a spore strain with an adjuvant effect that can efficiently activate CD8+ T cells is obtained to make up for the deficiency of a simple gene vaccine; the fusion model gene Sj26GST is designed by using immune bioinformatics and structural biology techniques -TTFC, and Sj26GST-TTFC was fused and expressed in the constructed IL-2 spore strain through the capsid protein CotC, and the CotB-IL-2/CotC-Sj26GST-TTFC epitope double transposition recombinant Bacillus subtilis vaccine strain was constructed. The constructed recombinant bivalent-IL-2-Bacillus subtilis vaccine strain has fast production and low cost, and can be used for mucosal immunity through nasal drops or nasopharyngeal spray, laying a technical and theoretical foundation for the development of bivalent vaccines with lasting immune protection.

The beneficial effects of the present invention are:

The invention is based on the good research foundation of the invention team in immunology, pathogenic biology, bioinformatics, molecular biology, epidemiology and nano-biotechnology, combined with project team members to deliver the vaccine carrier and adjuvant in the Bacillus subtilis mucosa Important progress has been made in research, model antigen research, and bivalent vaccine research, and systematically carry out basic research on the application of Bacillus subtilis mucosal delivery carrier technology to bivalent vaccines. On the basis of the fusion expression of bivalent exogenous antigen proposed by the present invention, the international mature Bacillus subtilis spore mucosal immune delivery carrier vaccine research technology is combined with the bivalent fusion expression pattern antigen gene technology, and the cytokine IL-2 is used as an immune regulator The adjuvant molecule can realize local mucosal immunity in the skin and mucous membrane of the pathogenic microorganism invasion portal, further activate the systemic immune response, especially the specific CD8+ T cell immune response, and achieve the effect of cross-immune protection. Bacillus subtilis is easy to genetically modify, and the production conditions and equipment are simple. The spore vaccine is suitable for safe, efficient, and economical mass production. It also has excellent thermal stability and long half-life. It is especially suitable for adding vaccines to animal feed on site and high-risk groups Intranasally or orally immunized, showing good dominance.

It has the following two aspects of innovation and advantages:

(1) Joint integration and innovation of multiple technologies such as Sj26GST-TTFC bivalent fusion expression antigen sequence, cytokine adjuvant IL-2 and Bacillus subtilis spore mucosal vaccine carrier technology. The spore vaccine carrier technology realizes the adjuvant effect of nano-scale biological carriers on the delivery of antigens, realizes oral or nasal mucosal immunization, and ensures the high efficiency of subsequent vaccine production and the low cost of storage and transportation; the cytokine adjuvant IL-2 The introduction of the modulator makes it possible to adjust the type of immunity, which promotes the enhancement of specific CD8+ T cell immune response. This project introduces the joint integration innovation of multiple technologies, forms an organic whole with complementary advantages, and improves the new technology theory of mucosal immune vaccine. The research in this aspect is also a theoretical innovation.

(2) Innovative design of adjuvants. This project takes advantage of the immune-enhancing adjuvant effect of the new microbial carrier and the immune-modulating adjuvant effect of the cytokine IL-2. Combining the advantages of the two can improve the immune protection effect of the vaccine, significantly reduce the dose of the vaccine and reduce the immune response. The number of times provides a guarantee for the immunization of feed-added vaccines and special populations.

The present invention has the following three characteristics:

(1) Facing the urgent needs of public health, the development of highly effective oral probiotic-type Schistosomiasis japonicum vaccine and tetanus vaccine is an effective way to prevent the transmission and prevalence of Schistosomiasis japonicum and tetanus. The introduction of multiple vaccine development technologies and the integrated innovation of new adjuvant technologies is an applied basic research that highlights the combination of scientific theory and practical application, and can solve practical problems for the country;

(2) Bivalent oral or intranasal vaccines can improve the immunity of the population, which is in line with the concept of One Health. Vaccine research and development technology can be applied to influenza or bird flu vaccines. For high-risk groups, use nasopharyngeal spray mucosal immunization to achieve occupational protection and immunity for high-risk groups including poultry workers. Improving group immunity is also a way to block new outbreaks of human, animal and poultry Effective means of transmission of influenza to the general population.

(3) Bacillus subtilis spore-carrier mucosal vaccine has good thermal stability. The construction method of recombinant spores can protect exogenous antigens and prolong the half-life of the vaccine. It is easy to store and transport at room temperature, and is suitable for developing countries and underdeveloped regions. Compared with the traditional vaccine development method, it is a breakthrough and has huge research and development prospects and commercial application value.

Description of drawings

Figure 1 shows the cell division and spore formation process of Bacillus subtilis B.subtilis (F, forespore endophytic spore; MC, mother cell mother cell; S, spore spore; VC, vegetative cell propagating body cell.);

Figure 2 is the electron microscope image and structural distribution diagram of B. subtilis Spore capsid protein (Daisuke etc, Yakugaku zasshi, 2012);

Figure 3 is a schematic diagram of the construction of the cotB-Peptide linker-IL-2 fusion gene in Example 1 of the present invention;

Figure 4 is a schematic diagram of the construction of the cotC-Sj26GST-TTFC fusion gene in Example 1 of the present invention;

Fig. 5 is a schematic diagram of the fusion expression of CotB-IL-2 in the outermost layer of the spore capsid in Example 1 of the present invention;

Fig. 6 is a schematic diagram of fusion expression of CotC-Sj26GST-TTFC epitope peptide in the outermost layer of spore capsid in Example 1 of the present invention;

Fig. 7 is a schematic diagram of transposition of two exogenous genes to the chromosome of Bacillus subtilis in Example 1 of the present invention;

Figure 8 is a schematic diagram of simultaneous double fusion expression of CotB-IL-2 and CotC-Sj26GST-TTFC epitope peptide in the outermost layer of spore capsid in Example 1 of the present invention;

Figure 9 is the PCR identification of Sj26GST, TTFC and Sj26GST-TTFC positive clones in Example 1 of the present invention, and the first to fourth lanes correspond to the PCR products of Marker DL10000, Sj26GST, TTFC and Sj26GST-TTFC respectively;

Figure 10 is the PCR identification of recombinant spore positive clones in Example 1 of the present invention;

Figure 11 is the expression figure of SDS-PAGE and Western blot analysis SjGST, TTFC and SjGST-TTFC protein in Example 1 of the present invention; Figure A, 12% SDS-PAGE. Analysis of the expression of three proteins. When IPTG induced expression ( lane3), the target proteins (SjGST, TTFC and SjGST-TTFC) were all significantly expressed, and the non-induced group was used as the control (lane 4). Lane 2 was the purified SjGST, TTFC and SjGST-TTFC proteins (pointed by the arrow). Figure B ,Western blot proved the protein expression. The primary antibodies incubated with Sj26GST, TTFC, and Sj26GST-TTFC were mouse anti-Sj26GST serum, mouse anti-TTFC serum, and mouse anti-Sj26GST-TTFC serum, respectively. HRP goat anti-mouse antibody was used as the secondary antibody. The protein size indicated by the arrow;

Figure 12 shows the detection of the expression of Sj26GST-TTFC in the spore coat by immunofluorescence and laser confocal microscopy in Example 1 of the present invention. The expression of the CotC-Sj26GST-TTFC recombinant spore strain shown in panel A. Samples were incubated with purified rabbit anti-TTFC serum, and the secondary antibody was incubated with anti-rabit.IgG-Fluoresceinisothiocyanate (green, FITC; sigma); the recombinant CotC-Sj26GST-TTFC spores shown in Figure B were incubated with mouse anti-Sj26GST antibody, and the secondary antibody was anti-mouse IgG-tetramethyl rhodamine isothiocyanate conjugate (red, TRITC; Sigma). The non-recombinant spore wild strain PY79 shown in Figure C was used as a control;

Figure 13 shows the expression of IL-12 detected on the surface of recombinant spores by immunofluorescence and laser confocal microscopy in Example 1 of the present invention; the sample was incubated with purified mouse anti-IL-2 serum, and the secondary antibody was incubated with anti-mouse anti-mouseIgG- tetramethylrhodamine isothiocyanate conjugate (red, TRITC; Sigma). The expression of the recombinant spore strain cotB-IL-2 on the spore surface shown in Figure a; the non-recombined wild strain PY79 shown in Figure b was used as a control;

Figure 14 is the level change of anti-Sj26GST-TTFC specific serum IgG after mucosal immunization in Example 1 of the present invention;

Fig. 15 Detection of whole spleen cell IFN-γ in Example 1 of the present invention;

Figure 16 is the immunofluorescence and flow cytometry detection in Example 1 of the present invention;

Expression of pUS186-CotC-Sj26GST on the surface of spores;

Fig. 17 shows the level of serum Sj26GST-specific IgG, the ratio of IgG1/IgG2a and the level of fecal sIgA (OD ₄₅₀ ) after oral administration of recombinant spore WB600/pUS186-CotC-GST in Example 1 of the present invention to immunize mice.

detailed description

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples.

Example 1

The inventors of the present application used the Schistosoma japonicum 26kDa GST (Sj26GST) protein as a model antigen in the previous research, and used the Bacillus subtilis expression plasmid pUS186 to construct the Sj26GST gene in the downstream of the Bacillus subtilis capsid gene CotC promoter and its coding sequence, at WB600 The exogenous protein Sj26GST was highly expressed on the surface of spore capsid in the extracellular enzyme-deficient strain.

Using the Bacillus subtilis shuttle integration plasmid pDG1664, CotC-Sj26GST-TTFC was integrated into the chromosome of Bacillus subtilis through double crossover replacement, and fused with CotC to express on the outer surface of the spore capsid; at the same time, the CotB-peptide linker was integrated using the Bacillus subtilis shuttle integration plasmid pDG364 - IL-2 is integrated into the chromosome of Bacillus subtilis through double crossover replacement, and is fused with CotB to express on the outer surface of the spore capsid, realizing the construction of a multivalent antigen fusion expression system using endophytic spores of Bacillus subtilis. Fluorescence immunology experiments and laser confocal microscopy detected that foreign proteins were successfully expressed on the outer surface of spores (Figure 13), and also proved that the macromolecular structure of the multivalent recombinant antigen did not affect the assembly of spore outer layer proteins and spore formation.

To evaluate the immune adjuvant effect of spores, we used molecular informatics to analyze and experimentally detected the physicochemical properties of different species of Bacillus, Bacillus subtilis PY79, HU58 and Bacillus clausii O/C were selected as non-recombinant The carrier of the spore-protein vaccine was mixed with the protein, which confirmed the adsorption of the spores to the protein. Immunizing the mice with the spores adsorbed by the protein can cause a strong mucosal immune response, and high levels can be detected in lung tissue, feces, and saliva. Levels of Sj26GST-specific sIgA secretion. At the same time, it can also activate the systemic immune response in mice, and the IgG subclass suggests a Th1/Th2 mixed immune response dominated by Th1. It has been confirmed that spores, as a new type of mucosal vaccine adjuvant, can improve the body's specific and non-specific immune levels, and regulate the type of immune response.

Studies have shown that the Bacillus subtilis spore vaccine platform can induce mucosal and systemic immune responses against specific pathogens, including the immune response of specific CD8+ T lymphocytes, which can effectively prevent the infection of pathogenic organisms and provide a theoretical basis for the development of mucosal vaccines.

1. Research objectives

In this paper, a multivalent antigen fusion expression system was constructed using endophytic spores of Bacillus subtilis. Schistosoma japonicum 26kDa GST (Sj26GST) protein was selected as the model antigen and tetanus toxin C fragment (TTFC) was used as the immune adjuvant molecule, which was displayed on the outer surface of the spore (with The spore CotC coat protein is fused to express CotC-Sj26GST-TTFC); and the construction is combined with the double transfection construction for displaying IL-2 on the outer surface of the spore (expressed in fusion with the spore CotB-IL-2 coat protein). Fluorescence immunology experiments and laser confocal microscopy detected that foreign proteins were successfully expressed on the outer surface of spores, which also proved that the macromolecular structure of multivalent recombinant antigens did not affect the assembly of spore outer layer proteins and spore formation, and proved that IL- 2 can improve the level of cellular immunity of the vaccine.

2. Research content

(1) Bioinformatics analysis and gene fusion of bivalent pattern antigen gene sequence

1) Using Schistosoma japonicum 26kDa GST (Sj26GST) protein as the model antigen and tetanus toxin C fragment (TTFC) as the immune adjuvant molecule, analyzing its gene sequence, and recombining the fusion Sj26GST-TTFC gene sequence

2) Recombinant sequence sequencing detection

3) Construct the recombinant sequence Sj26GST-TTFC at the C-terminus of the spore capsid protein CotC gene

4) Fusion expression of CotC-Sj26GST-TTFC on the capsid surface of Bacillus subtilis and identification of fusion expression

(2) Construction of IL-2 expressing spores with adjuvant effect

1) Analyze the sequence of cytokine IL-2 and spore capsid CotB

2) Construction of IL-2 at the C-terminus of the spore capsid protein CotB gene by connecting fragments

3) Fusion expression of CotB-IL-2 on the capsid surface of Bacillus subtilis and identification of fusion expression

(3) Construction of recombinant Bacillus subtilis vaccine IL-2 and Sj26GST-TTFC double expression vaccine strain

1) Analysis of spore capsid CotC sequence, analysis of recombinant fusion gene Sj26GST-TTFC sequence

2) Construct the fusion Sj26GST-TTFC sequence at the C-terminus of the CotC gene

3) On the basis of IL-2 expressing spores, construct the Sj26GST-TTFC spore capsid fusion double expression strain, namely CotB-IL-2/CotC-Sj26GST-TTFC

(4) Biological activity identification and protective evaluation of Bacillus subtilis spore vaccine

1) Safety determination of recombinant or non-recombinant vaccines

2) Research on immunological characteristics of vaccines and determination of immune levels

3) Experimental research on the protective attack of vaccines

3. Research methods and experimental means

(1) Construction of recombinant Bacillus subtilis vaccine candidate strain

1) Construction of amyE:cotB-IL-2 recombinant Bacillus subtilis shuttle integration plasmid

A 1091bp DNA fragment of cotB promoter and coding sequence was amplified from B. subtilis PY79 chromosome and inserted into plasmid pDG364. pDG364 carries a resistance cat gene and a multiple cloning site at the site of amylase amylase (amyE), and can integrate the carried cat and inserted foreign fragments into the chromosome of B. subtilis through the amyE gene. At the downstream C-terminus of pDG364-cotB, a total of 457bp of artificially synthesized sequences were cloned: including the peptide linker sequence GGGEAAAKGGG (peptide linker) (SEQ ID NO.1) sequence with a strong α-helical structure and human IL- derived from Homo sapiens 2 sequence, the constructed plasmid is pDG364-cotB-linker-IL2, and sequenced to identify whether the construction is successful (as shown in Figure 3).

The gene sequence of the peptide link of the alpha helix structure is directly synthesized in the primer:

linker-gggeaaakggg-F (with Eco RI):

GCC TGT TAG GAA TTC CGC TCC AAT CTC TTT TTA CAA TAG AAT ATA TGG AACCGA AAA TCA TGG CGA TGT ATG AAC GGA TTA GGC C

linker-gggeaaakggg-R (with Bam HI):

CGC GGA TCC TCC TCC TCC ACC TTT CGC TGC TGC TTC TCC TCC ACC GGA TGA TTGATC ATC TGA AG

In the cotB-linker-IL2 sequence, the primer sequence of IL2:

IL2link-F (with Pcil):

5'-GCT TC A CAT GT T TAC GTC AGT GTA GAG ATG ATA GAT TGG C-3'

IL2link-R (with BamHI):

5'-CAT AT G GAT CC G GTG GAG GAG AAG CAG CAG CG-3'.

2) construction of thrC:cotC-Sj26GST-TTFC recombinant Bacillus subtilis shuttle integration plasmid

The cotC promoter and coding sequence were amplified from B. subtilis PY79 chromosome and inserted into pDG1664. pDG1664 carries the ErmR gene and multiple cloning sites at the thrC gene site, and can integrate the carried ErmR and inserted foreign fragments into the B. subtilis chromosome through the thrC gene. In the second step, the designed and synthesized Sj26GST-TTFC sequence was amplified and inserted into the downstream C-terminus of pDG1664-CotC. The constructed recombinant plasmid was pDG1664-cotC-Sj26GST-TTFC, and sequenced to verify whether the construction was successful (as shown in Figure 4).

Table 1 Gene amplification primers

Sj26GST contains a 657bp longest open reading frame, the start codon is ATG, the stop codon is TGA, encoding 218 amino acids. Its theoretical molecular weight and isoelectric point are 25.63kDa and 6.09, respectively. Its nucleotide sequence is shown in SEQ ID NO:2, and its amino acid sequence is shown in SEQ ID NO:3.

TTFC contains a longest open reading frame of 1359bp, the start codon is ATG, the stop codon is TAA, encoding 452 amino acids. Its theoretical molecular weight and isoelectric point are 51.77kDa and 6.7, respectively. Its nucleotide sequence is shown in SEQ ID NO:4, and its amino acid sequence is shown in SEQ ID NO:5.

Sj26GST-TTFC contains a 2019bp longest open reading frame, the start codon is ATG, the stop codon is TAA, encoding 672 amino acids. Its theoretical molecular weight and isoelectric point are 77.4kDa and 6.27, respectively. Its nucleotide sequence is shown in SEQ ID NO:6, and its amino acid sequence is shown in SEQ ID NO:7.

The Sj26GST sequence obtained by PCR of Sj26GST-P1 and Sj26GST-P5 can be linked with the TTFC sequence obtained by PCR of TTFC-P3 and TTFC-P4 primers to form a Sj26GST-TTFC sequence, and the corresponding restriction site can be inserted just downstream of the cotC sequence.

The Sj26GST sequence from Sj26GST-P1 and Sj26GST-P2 PCR was used to insert into PET vector alone, expressing GST protein for antibody production.

3) Construction of fusion expression of IL-2 and Bacillus subtilis capsid CotB

The recombinant plasmid pDG364-cotB-linker-IL2 was linearized by single enzyme digestion outside the insert fragment, and transformed into PY79 competent cells, and the positive clones were selected from the negative transformants in the amylase plate test with CmR (5 μg/ml) LB plates, and named It is IL-2 Bacillus subtilis strain, which will express CotB-linker-IL-2 fusion protein. The fusion expression protein was identified and quantitatively analyzed by flow cytometry, Western Blot and Dot Blot, and the fusion protein was expressed in the outermost layer of the spore by immunofluorescence and laser confocal microscopy (as shown in Figure 5).

4) Construction of fusion expression of Sj26GST-TTFC and Bacillus subtilis capsid CotC

The recombinant plasmid pDG1664-cotC-Sj26GST-TTFC was linearized by single enzyme digestion outside the insert fragment and transformed into PY79 competent cells, and the ThrC-negative transformants were screened with ErmR (1 μg/ml) LB plate to select positive clones, named Sj26GST-TTFC subtilis strain, this strain will express CotC-Sj26GST-TTFC fusion protein. The fusion expression protein was identified and quantitatively analyzed by flow cytometry, WesternBlot and Dot Blot, and the fusion protein was expressed in the outermost layer of the spore by immunofluorescence and laser confocal microscopy (as shown in Figure 6).

5) Construction of dual expression strains of IL-2 and Sj26GST-TTFC (amyE::cotB-IL2; thrC::cotC-Sj26GST-TTFC)

The chromosome of the recombinant strain Sj26GST-TTFC subtilis spore constructed above was extracted, transformed into competent cells produced by IL-2 recombinant spore strain, and positive clones were screened with ErmR (1 μg/ml). The double-transposition recombinant spore strain will simultaneously express two fusion proteins of CotB-IL2 and CotC-Sj26GST-TTFC in the outermost layer of the spore capsid (as shown in Figures 7 and 8). The fusion protein was identified and quantitatively analyzed by flow cytometry, Western Blot and Dot Blot, and the fusion protein was expressed in the outermost layer of spores by immunofluorescence and laser confocal microscopy.

(4) Safety determination of recombinant spore vaccine

Experimental animals and grouping: sterile Balb/c mice, male and female 6-8w, 20 in each group, set up recombinant spore candidate vaccine group A, non-recombinant spore candidate vaccine group B, blank control group, a total of three groups. The rats were inoculated with the candidate spore vaccine by intranasal drops, and the blank control group did not receive any treatment. In the following 48 hours, the temperature changes, activity levels, food intake and drinking times of the rats in each group were closely observed and recorded. On the 3rd, 7th, and 21st days after immunization, three mice were randomly sacrificed respectively, and tissues such as muscle, heart, liver, spleen, lung, kidney, and brain at the injection site were taken, fixed in 4% paraformaldehyde, and paraffin sections were made, and microscope Next, observe whether there are pathological changes in the organs and tissues. After 8 weeks of immunization, the test mice were mixed with male and female and allowed to mate naturally. After birth, count and observe the number of pups, deformed pups, number of deaths after birth and development status.

(5) Research on the immunological characteristics of vaccines

1) Evaluation of mucosal immune effect of recombinant spore vaccine

Experimental animals and grouping: sterile Balb/c mice, female 6-8w, 6 in each group, set up Naive group, PY79, CotB-IL-2 spore group, CotC-Sj26GST-TTFC spore group, CotB-IL-2 /CotC-Sj26GST-TTFC double-expression spore group, 2μg IL-2+2μg Sj26GST-TTFC nasal drop group, 2μgSj26GST-TTFC nasal drop group and other 7 groups. Mice were lightly anesthetized with halothane by an anesthesia machine, and were inoculated with 2×10 ⁹ spores on the 1st, 22nd, and 43rd day with nasal drops (Gilson pipette tip, 40 μl). Serum, saliva, and feces were collected at -1, 20, 41, and 62 days and stored at -20°C for later use. On the 62nd day after the last immunization, the mice were sacrificed with CO2, and the blood was obtained by sticking a needle outside the chest cavity of the heart. ELISA detection of serum specific IgG and subclasses and specific sIgA levels of saliva, feces, and lung tissue extracts; after spleen cells were cultured, cellular immunity level analysis: measuring the percentage of CD4+ and CD8+ cells in spleen cells, and detecting the spleen after antigen stimulation The levels of cytokines such as IFN-γ and TNF-α secreted by the cells, and the proliferation of lymphocytes were detected by MTT.

3. Research results

(1) Identification of recombinant pET-28b-SjGST-TTFC plasmid

Pick the transformed single clone to extract the plasmid, and use the recombinant plasmid as a template to perform PCR with SjGST, TTFC, and SjGST-TTFC specific primers, and all fragments with the same size as the target gene are obtained (Figure 9). The sequencing results of the positive clones showed that the inserted fragments were completely consistent with the gene sequences of SjGST, TTFC, and SjGST-TTFC, indicating that the recombinant protein and fusion protein genes were successfully cloned.

(2) PCR identification of positive clones of recombinant spores

Figure 10 shows the recombinant Bacillus subtilis spores cultured after the get, tetC, and gst-tetC genes were cloned into the Bacillus subtilis gene, and colony PCR detection was performed directly to determine whether the clone was successful. In international practice, lowercase represents the gene name, for example tetC represents the gene name, and TTFC capitalizes the corresponding protein writing.

(3) WB analysis of recombinant Sj26GST, TTFC, SjGST-TTFC protein immunogenicity

Using the prepared anti-Sj26GST, anti-TTFC, and anti-SjGST-TTFC mouse-specific antisera, Western blot analysis showed that the three recombinant proteins could be recognized by specific antiserum, indicating that the Sj26GST, TTFC, and SjGST-TTFC recombinant proteins had immunogenic properties. sex and immunoreactivity (Figure 11).

(4) Immunofluorescence and laser confocal microscopy analysis of the display of Sj26GST-TTFC on the surface of recombinant spores

As shown in Figure 12, immunofluorescence and laser confocal microscopy showed that the CotC-Sj26GST-TTFC recombinant spores were successfully displayed on the spore surface through the spore coat protein CotC.

(5) Immunofluorescence and laser confocal microscopy analysis of the display of IL-2 on the surface of recombinant spore CotB-IL-2

As shown in Figure 13, immunofluorescence and confocal microscopy showed the successful expression of CotB-IL-2 on the surface of spores.

(6) Changes in the level of anti-Sj26GST-TTFC-specific serum IgG after mucosal immunization

Mice in each group were immunized with intranasal drops after mild anesthesia. The results shown in Figure 14 clearly show that the CotB-IL2/CotC-Sj26GST-TTFC recombinant spore group detected significantly higher serum IgG levels on the 21st day and 42nd day than the control group rSj26GST-TTFC group, PY79 group and Naive group (P< 0.05),

(7) The level of IFN-γ secreted by splenocytes

To evaluate the cellular immune response induced by recombinant spore vaccine immunization in mice, we detected the level of IFN-γ secreted by splenocytes. The data shown in Figure 15 shows that the IFN-γ level of the CotB-IL2/CotC-Sj26GST-TTFC recombinant spore nasal drop group (P<0.05) was significantly higher than that of the control group, which was statistically significant.

(8) Immunofluorescence and flow cytometry to detect the expression of pUS186-CotC-Sj26GST on the surface of spores

For the CotC-Sj26GST successfully expressed in the spore strain WB600 in the present invention using the pUS186 vector, the expression of GST protein on the outer surface of the spore was detected by flow cytometry and immunofluorescence microscopy (as shown in FIG. 16 ).

(9) Serum Sj26GST-specific IgG level, IgG1/IgG2a ratio and fecal sIgA level (OD ₄₅₀ ) after intragastric administration of recombinant spore WB600/pUS186-CotC-GST to mice

After oral immunization of mice with recombinant spore CotC-GST, specific anti-SjGST secreted IgA (sIgA) could be detected in the feces extract, as shown in b in Figure 17 above. The results showed that the specific sIgA level in the feces of the CotC-SjGST recombinant spore immunized group was significantly higher than that of the control group (CotC spore group and naive group, P<0.05). On the 16th day, the sIgA could be detected to be significantly increased, and reached the peak on the 33rd day, but the fecal sIgA was significantly lowered on the 51st day compared with the 33rd day (P<0.05). The mice were immunized orally, and the serum specific anti-SjGST total IgG antibody level was detected (a in Fig. 17). The results showed that on the 33rd day after the second immunization and on the 51st day after the third immunization, the CotC-SjGST recombinant spore immune group specifically IgG was significantly higher than that in the control group, P<0.05 (CotC spore immunization group and naive group). There was no significant difference between CotC spore immunization group and naive group, P>0.05. The results showed that Sj26GST was fused with CotC on the capsid surface of Bacillus subtilis WB600 and was immunogenic, which could stimulate the body to generate systemic immune response. To further analyze the IgG subtype level, the results of detecting Sj26GST-specific IgG1 and IgG2a showed that both IgG1 and IgG2a increased on the 33rd day after immunization, and continued to increase until the 51st day (c in Figure 17). On the 16th day, the IgG1 antibody level was 1.19±0.19 times higher than the IgG2a antibody level, but there was no significant difference (P>0.05). After the second immunization, the IgG2a antibody level increased 1.28±0.16 times higher than the IgG1 antibody level (P>0.05), after the last immunization, on the 51st day, the detection of IgG2a continued to increase to 1.52±0.23 times the level of IgG1 (P>0.05). It shows that after the first immunization, the Th2-type immune response is dominant, while after the second and third immunization, the Th1-type immune response is dominant, but there is no significant statistical difference, suggesting that the CotC-Sj26GST recombinant spore vaccine orally immunized Mice may have a Th1/Th2 mixed immune response.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

sequence listing

<110> Sun Yat-sen University

<120> A bivalent oral or nasal vaccine against Schistosoma japonicum and tetanus

<160> 16

<170> PatentIn version 3.3

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Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

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Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

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Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

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Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

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Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

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Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

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Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

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Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

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Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

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Thr Phe Gly Gly Gly Asp His Pro Pro Lys

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atatattata gaaggttata taatggacta aaattttatta taaaaagata tacacctaat 960

aatgaaatag attcttttgt taaatcaggt gattttatta aattatatgt atcatataac 1020

aataatgagc aattgtagg ttatccgaaa gatggaaatg cctttaataa tcttgataga 1080

attctaagag taggttataa tgccccaggt atccctcttt ataaaaaaat ggaagcagta 1140

aaattgcgtg atttaaaaac ctattctgta caacttaaat tatatgatga taaaaatgca 1200

tctttaggac tagtaggtac ccataatggt caaataggca acgatccaaa tagggatata 1260

ttaattgcaa gcaactggta ctttaatcat ttaaaagata aaattttagg atgtgattgg 1320

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Met Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val

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Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile

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Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp

35 40 45

Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn

50 55 60

Asn Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr

65 70 75 80

Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro

85 90 95

Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser

100 105 110

Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp

115 120 125

Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser

130 135 140

Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe

145 150 155 160

Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp

165 170 175

Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser

180 185 190

Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr

195 200 205

Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp

210 215 220

Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys

225 230 235 240

Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly

245 250 255

Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser

260 265 270

Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu

275 280 285

Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg

290 295 300

Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn

305 310 315 320

Asn Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr

325 330 335

Val Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly

340 345 350

Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala

355 360 365

Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp

370 375 380

Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala

385 390 395 400

Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro

405 410 415

Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys

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Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly

435 440 445

Trp Thr Asn Asp

450

<210> 6

<211> 2019

<212>DNA

<213> Artificial sequence

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tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

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aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaaggatcc 660

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agtacaattt taaatttaga tattaataat gatattatat cagatatatc tgggtttaat 780

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catttagtaa acaatgaatc ttctgaagtt atagtgcata aagctatgga tattgaatat 900

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agtctatcaa taggatctgg ttggagtgta tcacttaaag gtaataactt aatatggact 1080

ttaaaagatt ccgcggggaga agttagacaa ataactttta gggatttacc tgataaattt 1140

aatgcttatt tagcaaataa atgggttttt ataactatta ctaatgatag attatcttct 1200

gctaatttgt atataaatgg agtacttatg ggaagtgcag aaattactgg tttaggagct 1260

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ttatacacaa gttattttc tataaccttt ttaagagact tctggggaaa ccctttacga 1440

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aatataacag attatatgta tttgacaaat gcgccatcgt atactaacgg aaaattgaat 1560

atatattata gaaggttata taatggacta aaattttatta taaaaagata tacacctaat 1620

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aataatgagc aattgtagg ttatccgaaa gatggaaatg cctttaataa tcttgataga 1740

attctaagag taggttataa tgccccaggt atccctcttt ataaaaaaat ggaagcagta 1800

aaattgcgtg atttaaaaac ctattctgta caacttaaat tatatgatga taaaaatgca 1860

tctttaggac tagtaggtac ccataatggt caaataggca acgatccaaa tagggatata 1920

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tactttgtac ctacagatga aggatggaca aatgattaa 2019

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<213> Artificial sequence

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Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Gly Ser Met Lys Asn Leu

210 215 220

Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu Lys Lys

225 230 235 240

Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile

245 250 255

Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln Leu Val

260 265 270

Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu Ser Ser

275 280 285

Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe

290 295 300

Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala

305 310 315 320

Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser

325 330 335

Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu

340 345 350

Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu Val

355 360 365

Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu

370 375 380

Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser

385 390 395 400

Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala Glu Ile Thr

405 410 415

Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp

420 425 430

Arg Cys Asn Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile

435 440 445

Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser

450 455 460

Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg

465 470 475 480

Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp

485 490 495

Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro

500 505 510

Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn

515 520 525

Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp

530 535 540

Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn

545 550 555 560

Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn

565 570 575

Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro

580 585 590

Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr

595 600 605

Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu

610 615 620

Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile

625 630 635 640

Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu

645 650 655

Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp

660 665 670

<210> 8

<211> 85

<212>DNA

<213> Artificial sequence

<400> 8

gcctgttagg aattccgctc caatctcttt ttacaataga atatatggaa ccgaaaatca 60

tggcgatgta tgaacggatt aggcc 85

<210> 9

<211> 62

<212>DNA

<213> Artificial sequence

<400> 9

cgcggatcct cctccacctt tcgctgctgc ttctcctcca ccggatgatt gatcatctga 60

ag 62

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<211> 40

<212>DNA

<213> Artificial sequence

<400> 10

gcttcacatg tttacgtcag tgtagagatg atagattggc 40

<210> 11

<211> 32

<212>DNA

<213> Artificial sequence

<400> 11

catatggatc cggtggagga gaagcagcag cg 32

<210> 12

<211> 24

<212>DNA

<213> Artificial sequence

<400> 12

cggctagcat gtcccctata ctag 24

<210> 13

<211> 24

<212>DNA

<213> Artificial sequence

<400> 13

cccaagcttt cattttggag gatg 24

<210> 14

<211> 28

<212>DNA

<213> Artificial sequence

<400> 14

cgcggatcca tgaaaaatct ggattgtt 28

<210> 15

<211> 29

<212>DNA

<213> Artificial sequence

<400> 15

cccaagcttt taatcatttg tccatcctt 29

<210> 16

<211> 23

<212>DNA

<213> Artificial sequence

<400> 16

cgcggatcct tttggaggat ggt 23

Claims (7)

1. a host bacterium, is characterized in that, described host bacterium is prepared by the following method: (1) The nucleotide sequence of the CotB gene promoter sequence, the CotB coding sequence, the linker and the nucleotide sequence of IL-2 are sequentially connected to the first expression vector to obtain the first expression vector of the cotB-linker-IL2 fusion protein Recombinant expression vector; (2) In the step (1), single enzyme digestion outside the insert fragment of the first recombinant expression vector makes the linearization of the first recombinant expression vector, and then transforms into the first Bacillus competent cell, and screens to obtain the expression of CotB-linker - the first positive strain of IL-2 fusion protein; (3) The nucleotide sequence of the CotC gene promoter sequence, the CotC coding sequence and the exogenous model gene Sj26GST-TTFC is sequentially connected to the second expression vector to obtain the second recombinant expression vector expressing the CotC-Sj26GST-TTFC fusion protein ; (4) In the step (3), single enzyme digestion outside the insert fragment of the second recombinant expression vector makes the linearization of the second recombinant expression vector, and then transforms into the second Bacillus competent cell, and screens to obtain the expression of CotC-Sj26GST - the second positive strain of the TTFC fusion protein; (5) Extract the chromosome of the second positive strain expressing the CotC-Sj26GST-TTFC fusion protein in the step (4), and transform it into the IL-2CotB-linker-IL-2 fusion protein expressed in the step (2). The competent cells prepared from the first positive strain were screened for positive clones expressing CotB-IL2 fusion protein and CotC-Sj26GST-TTFC fusion protein at the same time to obtain the host bacteria. 2. The host bacterium according to claim 1, characterized in that the amino acid sequence of the linker is as shown in SEQ ID NO.1. 3. The host bacterium according to claim 1, characterized in that the nucleotide sequence of Sj26GST-TTFC in the step (3) is as shown in SEQ ID NO.6. 4. The host bacterium according to claim 1, characterized in that the first expression vector in the step (1) is plasmid pDG364, and the second expression vector in the step (3) is plasmid pDG1664. 5. host bacterium according to claim 1, is characterized in that, in described step (2), the first bacillus competent cell is bacillus subtilis PY79 competent cell, second bacillus in described step (4) Competent cells are Bacillus subtilis PY79 competent cells. 6. The use of the host bacterium as claimed in claim 1 in the preparation of bivalent oral or nasal vaccines against Schistosoma japonicum and tetanus. 7. A bivalent oral or nasal vaccine of Schistosoma japonicum and tetanus, characterized in that, said bivalent oral or nasal vaccine of Schistosoma japonicum and tetanus contains a mixture cultured by the host bacterium described in any one of claims 1-4. The resulting spores.