Double-antigen type 1 diabetes mellitus vaccine targeting intestinal M cells and preparation method of double-antigen type 1 diabetes mellitus vaccine

Abstract

The invention relates to a double-antigen type 1 diabetes vaccine targeting intestinal M cells and a preparation method of the double-antigen type 1 diabetes vaccine, and belongs to the technical field of biology. The method comprises the following steps: connecting human GAD65191-320, INS17-110 and CKS9 targeting peptide CKSTHPLSC through G4S connecting peptide according to the sequence, optimizing lactobacillus codon preference, inserting the human GAD65191-320, the INS17-110 and the CKS9 targeting peptide CKSTHPLSC into a pNZ8148 vector together, transforming constructed recombinant plasmids into MC1061 competent escherichia coli for cloning amplification, and extracting the recombinant plasmids; the recombinant plasmid is electrically transferred into NZ9000 competent lactococcus lactis to construct a GAD65191-320 + INS17-110 + CKSTHPLSC recombinant lactic acid bacteria vaccine, and after amplification, Nisin is used for inducible expression of the fusion protein. According to the present invention, the recombinant lactic acid bacteria vaccine is orally taken to immunize STZ induced BALB / c diabetes model mice, such that the mice can be induced to produce the GAD65 + INS specific antibody, and the proliferation of regulatory T cells and dendritic cells secreting interleukin 10 can be promoted so as to induce the immune tolerance, inhibit the differentiation of young DCs and maintain the blood glucose level, such that the type 1 diabetes mellitus can be prevented and treated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a dual-antigen type 1 diabetes vaccine targeting intestinal M cells and a preparation method thereof. Background Art

[0002] Type 1 Diabetes (T1D) is an autoimmune disease that is common among adolescents. The effector T lymphocytes in the patient's body will mistakenly identify self-antigens and actively attack pancreatic β cells, leading to pancreatic inflammatory infiltration and necrosis, resulting in insufficient insulin secretion and chronic hyperglycemia. At present, the treatment of T1D is mainly based on insulin injection, combined with relatively conservative treatment methods such as diet and exercise. This method can only alleviate the patient's symptoms to a certain extent, but it cannot prevent and treat the disease. Long-term insulin injection not only brings huge economic pressure to patients, but also causes blood sugar instability after injection, which brings many inconveniences to life. The ultimate goal of studying the prevention or treatment of T1D is to prevent the body's own immune system from attacking pancreatic cells, reduce pancreatic inflammatory infiltration, protect existing functions, and promote pancreatic regeneration and repair. However, existing treatments cannot achieve this goal.

[0003] In the development of T1D patients, glutamic acid decarboxylase GAD65 and insulin INS are relatively important autoantigens. A large number of GAD65 antibodies and insulin antibodies can be detected in the serum of most patients. Glutamic acid decarboxylase GAD65 is composed of 585 amino acid residues. The expression level of glutamic acid decarboxylase GAD65 is highest in human pancreatic β cells. GAD65 peptides and plasmids can activate adaptive immune responses, so they are considered to be pancreatic antigens with strong immunogenicity.

[0004] Due to the adaptability of intestinal mucosal immunity, the mucosal immune response generated by orally delivered antigens into the intestine is more likely to cause specific immune unresponsiveness and low responsiveness, inducing immune tolerance. Using this mechanism, designing vaccines against self-antigens to induce protective immune tolerance, reshape the immune tolerance and immune regulation of the autoimmune system, and improve pathological immunity may be a new direction for the prevention and treatment of type 1 diabetes. When the antigens in the vaccine are successfully delivered to the intestine, they still face the problems of decreased immunogenicity, low expression dose, and difficulty in being taken up by presenting cells. In addition, M cells serve as the entrance for antigen uptake in the intestine, and targeting antigens to M cells can improve the efficiency of intestinal mucosal immunity. For vaccines against T1D, how to safely and effectively promote the differentiation of Treg cells, the proliferation of dendritic cells (DCs) that secrete interleukin 10, inhibit the proliferation of immature DC cells, and increase the survival rate of pancreatic islet cells may be the key issue for the success of the vaccine.

[0005] At present, most of the vaccines for T1D are single antigen vaccines without M cell targeting, which is not ideal for blood sugar control. This may be because the single antigen short peptide cannot induce sufficient immunogenicity or the single antigen cannot play a role in different stages of T1D, or the lack of targeting makes the antigen unable to be captured by M cells to induce mucosal immune response. Therefore, how to overcome the shortcomings of existing technologies is an urgent problem to be solved in the field of biotechnology. Summary of the invention

[0006] The purpose of the present invention is to solve the deficiencies of the prior art, make up for the deficiencies of the existing short peptide vaccines or single antigen vaccines, and provide a dual-antigen type 1 diabetes vaccine targeting intestinal M cells and a preparation method thereof. After immunization, the vaccine can induce the body to produce specific antibodies against GAD65+INS, and at the same time induce the body to produce immune tolerance, promote Treg cell differentiation, the proliferation of dendritic cells (DCs) that secrete interleukin 10, inhibit the proliferation of immature DC cells, lower blood sugar, and ultimately prevent or treat type 1 diabetes.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing a dual-antigen type 1 diabetes vaccine targeting M cells comprises the following steps: Step (1), the 191-320 amino acid sequence of the human glutamic acid decarboxylase 65 gene GAD65, the 17-110 amino acid nucleotide sequence of the insulin INS gene, and the nucleotide sequence of the CKS9 targeting peptide CKSTHPLSC are sequentially connected through the G4S connecting peptide GGGGS, and after the lactic acid bacteria codon preference is optimized, GAD65 191-320 +INS 17-110 +CKSTHPLSC gene fragment was inserted into pNZ8148 vector to construct GAD65 191-320+ INS 17-110+ CKSTHPLSC-pNZ8148 recombinant plasmid; Step (2), GAD65 constructed in step (1) 191-320+ INS 17-110+ The CKSTHPLSC-pNZ8148 recombinant plasmid was transformed into MC1061 competent Escherichia coli for cloning and amplification, and the recombinant plasmid was extracted; Step (3), the recombinant plasmid extracted in step (2) is electroporated into NZ9000 competent Lactococcus lactis to construct GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine; Step (4), GAD65 constructed in step (3) 191-320+ INS 17-110+After the CKSTHPLSC recombinant lactic acid bacteria vaccine was amplified, Nisin was used to induce the expression of fusion protein.

[0008] Furthermore, preferably, in step (1), the nucleotide sequence of amino acids 191-320 of the human glutamate decarboxylase 65 gene GAD65, amino acids 17-110 of the insulin INS gene, and the nucleotide sequence of the targeting peptide CKSTHPLSC are sequentially connected by a G4S connecting peptide GGGGS, and the GAD65 obtained by the connection is 191-320 +INS 17-110 +CKSTHPLSC nucleotide sequence 5' end with 6HIS tag, after lactic acid bacteria codon preference optimization, the final expression of GAD65 191-320+ INS 17-110+ The amino acid sequence of CKSTHPLSC is shown in SEQ ID NO.1; wherein: Among them, GAD65 191-320 The nucleotide sequence of the amino acid is shown in SEQ ID NO.2 after optimization of lactic acid bacteria codon preference; INS 17-110 The nucleotide sequence of the amino acid is shown in SEQ ID NO.3 after optimization of lactic acid bacteria codon preference; The nucleotide sequence of the targeting peptide CKSTHPLSC is shown in SEQ ID NO. 4 after optimization of lactic acid bacteria codon preference; The nucleotide sequence of the G4S connecting peptide GGGGS is shown in SEQ ID NO.5 after being optimized for lactic acid bacteria codon preference; The nucleotide sequence of the 6HIS tag is shown in SEQ ID NO.6.

[0009] Further, preferably, in step (1), the method of inserting the pNZ8148 vector is: Located in human GAD65 191-320 The 6His tag nucleotide sequence is directly inserted at the 5' end before the amino acid nucleotide sequence Nco I restriction site, directly inserted at the 3' end of the nucleotide sequence of the targeting peptide CKSTHPLSC XB I restriction site, and then insert Nco I restriction site and XB I restriction site nucleotide sequence and pNZ8148 vector, using Nco I enzyme, XB I enzyme double digestion, followed by T4 enzyme ligation overnight to construct the recombinant plasmid GAD65 191-320+ INS 17-110+ CKSTHPLSC-pNZ8148.

[0010] Furthermore, preferably, the specific method of step (2) is: (2.1) GAD65 191-320+ INS 17-110+ The CKSTHPLSC-pNZ8148 recombinant plasmid was transformed into MC1061 competent Escherichia coli, spread on LB solid medium plates containing chloramphenicol, and cultured inverted at 37°C overnight. Single colonies on the LB solid medium plates containing chloramphenicol were picked for positive clone identification; (2.2) Add the positive clones to a test tube containing LB liquid culture medium containing chloramphenicol and culture them in a constant temperature shaker at 37°C overnight. The next day, inoculate the bacterial liquid into LB liquid culture medium containing chloramphenicol and culture them in a constant temperature shaker at 37°C overnight. The next day, use a plasmid extraction kit to extract GAD65 from the bacterial liquid harvested from the clone amplification. 191-320+ INS 17-110+ CKSTHPLSC-pNZ8148 recombinant plasmid.

[0011] Furthermore, preferably, the rotation speed of the constant temperature shaker is 200 rpm / min.

[0012] Furthermore, preferably, the specific method of step (3) is: (3.1) Mix 40 μL NZ9000 competent Lactococcus lactis with 1 μL GAD65 191-320+ INS 17-110+ The CKSTHPLSC-pNZ8148 recombinant plasmids were evenly mixed and transferred into a 0.2 cm electroporation cuvette precooled at 4°C; (3.2) Set the electric shock parameters to 2000V, 25μF, 200Ω, and then place the conversion cup in the electric shock tank for electric shock; (3.3) After the electroporation, add 1 mL of GMMC recovery medium, place on ice for 5 min, transfer to a 1.5 mL Enpendof tube, and place in a 30°C incubator for 2 h to recover the culture solution; (3.4) Spread the recovered bacterial solution on GM17 solid medium containing 10 μg / mL chloramphenicol, invert it after the bacterial solution is completely absorbed, and incubate it at 30°C for 36 hours. When transformants appear, pick the positive clones; (3.5) The selected positive clones were inoculated into 5 mL of GM17 broth containing 10 μg / mL chloramphenicol to amplify GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine.

[0013] Further, preferably, in step (4), the specific method of inducing the expression of the fusion protein with Nisin is: (4.1) GAD65191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine was inoculated at a volume ratio of 1:25 between bacterial solution and GM17 broth containing 10 μg / mL chloramphenicol and cultured at 30°C until the bacterial OD 600 is 0.4-0.6, and the culture is obtained; (4.2) Add Nisin to the culture to a final concentration of 50 ng / mL and incubate at 30°C for 4 h to induce expression of the fusion protein; (4.3) Induce the successfully expressed GAD65 191-320+ INS 17-110+ The CKSTHPLSC recombinant lactic acid bacteria vaccine solution was centrifuged at 4°C, the precipitate was collected and diluted to 1×10 10 CFU / mL and stored at -40℃ until use.

[0014] The present invention also provides a dual-antigen type 1 diabetes vaccine targeting M cells prepared by the above-mentioned dual-antigen type 1 diabetes vaccine preparation method targeting M cells.

[0015] The present invention also provides GAD65 obtained by the preparation method of the above-mentioned dual antigen type 1 diabetes vaccine targeting M cells. 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine.

[0016] The present invention further provides the above-mentioned GAD65 191-320+ INS 17-110+ Application of CKSTHPLSC recombinant lactic acid bacteria vaccine in the preparation of dual-antigen type 1 diabetes vaccine targeting M cells.

[0017] The present invention selects multiple autoantigens to construct a vaccine and increases targeting, which can improve the efficiency of antigen delivery while broadening the vaccine protection spectrum and protecting patients at different stages.

[0018] The present invention uses lactic acid bacteria as a recombinant vaccine vector to construct a vaccine of GAD65+INS truncated optimized epitopes combined with targeting peptides, which has multiple advantages: (1) the culture operation is simple, the construction technology is mature, and the cost is low; (2) lactic acid bacteria can be selected for oral immunization, with high patient compliance, and is safer than attenuated vectors, and does not require subsequent treatment such as protein purification in vitro; (3) lactic acid bacteria have a strong adhesion effect on the body's mucosa and can survive in the mucosa after entering the intestine; (4) the recombinant lactic acid bacteria vaccine can release specific target antigen proteins to the body's M cells under the action of M cell targeting peptides, thereby improving the efficiency of antigen recognition; (5) it can directly stimulate mucosal immunity and systemic immunity; (6) lactic acid bacteria, as probiotics, can increase the stability of the intestinal flora structure, avoid intestinal flora disorders in T1D patients, regulate intestinal flora, and help prevent the progression of T1D. It makes up for the shortcomings of other vaccines such as intramuscular injection and complex purification of genetically engineered proteins, and is of great significance to the development of new vaccines.

[0019] The present invention proposes: the 191-320 amino acid sequence of the human glutamate decarboxylase 65 gene GAD65, the 17-110 amino acid nucleotide sequence of the insulin INS gene and the nucleotide sequence of the targeting peptide CKSTHPLSC are sequentially connected through the G4S connecting peptide GGGGS (i.e., sequentially connected in the above sequence order), and after the lactic acid bacteria codon preference is optimized, they are inserted into the pNZ8148 vector to construct GAD65 191-320+ INS 17-110+ CKSTHPLSC-pNZ8148 recombinant plasmid; transform the positive plasmid into NZ9000 competent Lactococcus lactis and induce the expression of fusion protein with 50ng / mL Nisin. The advantages of this construction method are: (1) The truncated and optimized GAD65 peptide and INS peptide contain specific antigenic epitopes of the two antigens while reducing the interference caused by nonspecific protein expression. In addition, they are combined with the targeting peptide CKSTHPLSC, which has a strong affinity for M cells. Compared with other monovalent vaccines, GAD65 191-320+ INS 17-110+ CKSTHPLSC covers a variety of antigenic epitopes and has M cell targeting; (2) Using lactic acid bacteria as a carrier can, to a certain extent, avoid the problem of antigenic peptides being degraded by enzymes in the gastrointestinal tract after oral administration, because the antigenic peptides can be directly delivered to the intestinal mucosa by lactic acid bacteria, stimulate mucosal immunity, and induce immune tolerance; (3) The lactic acid bacteria expression system has the advantages of simple construction, strong gene modification acceptance, and the ability to achieve specific expression of antigens in the body, and is a vaccine carrier with higher safety; the present invention uses GAD65 191-320+ INS 17-110+Oral immunization of BALB / c mice with the CKSTHPLSC recombinant lactic acid bacteria vaccine stimulates the production of specific IgG antibodies against GAD65 and INS. It also promotes the proliferation of Treg cells and dendritic cells (DCs) that secrete interleukin 10 in the body, thereby inducing immune tolerance, inhibiting the proliferation of immature DC cells, and maintaining blood sugar levels, thereby achieving the purpose of preventing and treating type 1 diabetes.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) Compared with existing monovalent vaccines, the recombinant lactic acid bacteria vaccine GAD65 191-320+ INS 17-110+ CKSTHPLSC covers a variety of antigenic epitopes and has M cell targeting; (2) Use the linker sequence to connect GAD65, INS protein and CKSTHPLSC targeting peptide respectively. The linker sequence is used to separate the proteins on both sides to allow them to complete their independent functions, ensuring the correct translation and folding of GAD65 and INS proteins and the exposure of their active sites; (3) The antigen is directly expressed in the intestinal mucosa by lactic acid bacteria, and has M cell targeting, and can be directly extracted by antigen presenting cells in the mucosal immune system, which to a certain extent avoids the degradation of the antigen when passing through the intestine. 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine, oral immunization of BALB / c mice (6-8 weeks old), oral administration of GAD65 191-320+ INS 17-110+ The number of lactic acid bacteria colonizing the intestine of BALB / c mice in the CKSTHPLSC (GIC) recombinant lactic acid bacteria vaccine group was compared with that of GAD65 prepared by the same method. 191-320+ INS 17-110 (GI) The lactic acid bacteria in the recombinant lactic acid bacteria and NZ9000 control group and Blank group increased by nearly 25 times; (4) Using streptozotocin (STZ), T1D model was established in the BALB / c mice that completed oral immunization at 13 weeks of age. The oral GIC recombinant lactic acid bacteria vaccine group produced GAD65 and INS specific IgG antibodies with a titer of 1:5 at 15 weeks of age; at 21 weeks of age, 57% of the mice still maintained the above antibody levels; (5) By measuring the blood glucose of mice modeled with T1D using STZ, it was found that the GIC group was able to alleviate the hyperglycemia of mice from the 21st to the 26th week of age; (6) By analyzing the islet area of ​​each group, there was no significant difference in the average area of ​​healthy islets between the oral GIC lactic acid bacteria vaccine group and the None group without STZ treatment, which can reduce islet damage during the development of T1D.

[0021] (7) GAD65 191-320+ INS 17-110+ The CKSTHPLSC recombinant lactic acid bacteria vaccine can significantly induce the proliferation of Treg cells and dendritic cells (DCs) secreting interleukin 10 in the body, inhibit the maturation of immature DC cells, and establish immune tolerance to two self-antigens, thereby alleviating type 1 diabetes.

[0022] The preparation method of the dual-antigen type 1 diabetes vaccine targeting M cells provided by the present invention is simple, utilizes the characteristics of oral immunization of lactic acid bacteria, has high compliance, and has M cell targeting, and has obvious immune tolerance effect. It can make up for the defects of existing monovalent vaccines such as single protection spectrum, complex protein purification process, and low induction efficiency, which lead to the inability to better induce Treg cells, and has broad development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 For GAD65 191-320+ INS 17-110+ Schematic diagram of the construction of the CKSTHPLSC-pNZ8148 recombinant plasmid; Figure 2 It is the colonization of lactic acid bacteria vaccine in the intestine 12 hours after oral immunization; Figure 3 The figure shows the changes in body weight and blood sugar of BALB / c mice aged 7-26 weeks; A shows the changes in body weight, and B shows the changes in blood sugar; Figure 4 The figure shows the detection of specific IgG antibodies of mouse GAD65 and INS serum; A is the result of serum collected at week 15, and B is the result of serum collected at week 21; Figure 5 The figure is a comparison of the total area and average area of ​​pancreatic islets in each group; A is the total area of ​​pancreatic islets in each group, and B is the average area of ​​pancreatic islets in each group; Figure 6 Figure 2 is a flow cytometric analysis of spleen and intestinal lymphocytes; A is the percentage of regulatory T cells (Treg) in spleen lymphocytes of each group, B is the percentage of regulatory T cells (Treg) in intestinal lamina propria lymphocytes of each group, C is the percentage of dendritic cells (DCs) secreting interleukin 10 in intestinal lamina propria lymphocytes of each group, and D is the percentage of immature DC cells in intestinal lamina propria lymphocytes of each group; In the figure: Blank is the group that only received STZ for T1D modeling; Control is the group that received oral lactic acid bacteria vaccine carrying pNZ8148 empty plasmid; GI group received oral GAD65 191-320+ INS 17-110 Recombinant lactic acid bacteria vaccine group; GIC group received oral GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine group; None group was the group without any treatment.

[0025] Note: Except for the None group, all BALB / c mice used in the present invention were subjected to T1D modeling using STZ at 13 weeks of age. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with embodiments.

[0027] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0028] The culture medium formula used in the present invention is as follows: GM17 broth containing 10μg / mL chloramphenicol: 37.3g / L M17 broth powder + 5g / L glucose + 10μg / mL chloramphenicol GM17 solid medium containing 10μg / mL chloramphenicol: 55g / L M17 agar powder + 5g / L glucose + 10μg / mL chloramphenicol GMMC recovery medium: GM17 medium + 5 g / L glucose + 20 mM MgCl2 + 2 mM CaCl2; LB solid medium plate containing chloramphenicol: 2g tryptone + 1g yeast powder + 2g NaCl + 4g agar dissolved in 200mL ultrapure water, sterilized at high temperature, cooled, and added with chloramphenicol at a final concentration of 10μg / mL; LB liquid culture medium containing chloramphenicol: 2 g tryptone + 1 g yeast powder + 2 g NaCl were dissolved in 200 mL ultrapure water, sterilized at high temperature, cooled, and chloramphenicol was added to a final concentration of 10 μg / mL. Example 1

[0029] A method for preparing a dual-antigen type 1 diabetes vaccine targeting M cells comprises the following steps: (1) The nucleotide sequence of amino acids 191 to 320 of the glutamate decarboxylase 65 gene GAD65, the nucleotide sequence of amino acids 17 to 110 of the insulin INS gene, and the nucleotide sequence of the targeting peptide CKSTHPLSC were connected in the above order through the G4S connecting peptide GGGGS; After connecting the linker sequence, a 6HIS tag was added to the 5' end of the nucleotide sequence and inserted into the pNZ8148 vector. After the lactic acid bacteria codon preference was optimized, the final GAD65 was expressed. 191-320 +INS 17-110 +CKSTHPLSC amino acid sequence is shown in SEQ ID NO.1; The GAD65 191-320 The nucleotide sequence of the amino acid is shown in SEQ ID NO.2 after optimization of lactic acid bacteria codon preference; The INS 17-110 The nucleotide sequence of the amino acid is shown in SEQ ID NO.3 after optimization of lactic acid bacteria codon preference; The nucleotide sequence of the targeting peptide CKSTHPLSC is shown in SEQ ID NO.4 after being optimized for lactic acid bacteria codon preference; The nucleotide sequence of the G4S connecting peptide GGGGS is shown in SEQ ID NO.5 after being optimized for lactic acid bacteria codon preference; The nucleotide sequence of the 6HIS tag is shown in SEQ ID NO.6; The original GAD65 gene sequence is shown in SEQ ID NO.7; The original INS gene sequence is shown in SEQ ID NO.8; GAD65 191-320 +INS 17-110 The theoretical molecular weight of the +CKSTHPLSC protein is: 27KDa; The method for inserting into pNZ8148 vector is: Located in human GAD65 191-320 The 6His tag nucleotide sequence is directly inserted at the 5' end before the amino acid nucleotide sequence Nco I restriction site (nucleotide sequence: ccatgg) was directly inserted at the 3' end of the nucleotide sequence of the targeting peptide CKSTHPLSC XB I restriction site (nucleotide sequence is tctaga), then insert Nco I restriction site and XB I restriction site nucleotide sequence and pNZ8148 vector, using Nco I enzyme, XB I enzyme double digestion, followed by T4 enzyme ligation overnight to construct the recombinant plasmid GAD65191-320 +INS 17-110 +CKSTHPLSC-pNZ8148; where: The enzyme digestion system is: 1 μL pNZ8148 plasmid, 1 μL Nco I enzyme, 1 μL XB I enzyme, 3 μL 10× Buffer, 24 μL water, a total of 30 μL, 37℃ digestion for one hour, purify DNA fragments according to the gel recovery kit to recover the digestion product; The insertion method is: GAD65 191-320 +INS 17-110 +CKSTHPLSC fragment with Nco I and XB I double enzyme digestion gel recovery after electrophoresis pNZ8148 vector fragments were ligated, the ligation reaction system is: 3μL GAD65 191-320 +INS 17-110 +CKSTHPLSC fragment, 1 μL pNZ8148 vector fragment, 2 μL 10× Buffer, 1 μL T4 DNA ligase, 3 μL water, a total of 10 μL, ligated at 16°C for 12 hours; (2) GAD65 191-320 +INS 17-110 The steps of transforming the +CKSTHPLSC-pNZ8148 recombinant plasmid into MC1061 competent E. coli for amplification, plasmid extraction, and screening of positive plasmids are as follows: (2.1) GAD65 191-320+ INS 17-110+ The CKSTHPLSC-pNZ8148 recombinant plasmid was transformed into MC1061 competent Escherichia coli, spread on LB solid medium plates containing chloramphenicol, and cultured inverted at 37°C overnight. Single colonies on the LB solid medium plates containing chloramphenicol were picked for positive clone identification; (2.2) Add the positive clones to a test tube containing 5 mL of LB liquid medium containing chloramphenicol and culture them overnight in a constant temperature shaker at 37°C and 200 rpm / min. The next day, inoculate the 5 mL of bacterial culture into 400 mL of LB liquid medium containing chloramphenicol and culture them overnight in a constant temperature shaker at 37°C and 200 rpm / min. The next day, use a plasmid extraction kit (Wizard® Plus SV minipreps DNA Purification Systems, Promega) to extract GAD65 from the bacterial culture harvested from the clone amplification. 191-320+ INS 17-110+ CKSTHPLSC-pNZ8148 recombinant plasmid; (3) The recombinant plasmid extracted in step (2) was electroporated into NZ9000 competent Lactococcus lactis to construct GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine; The specific method is: (3.1) Mix 40 μL NZ9000 competent Lactococcus lactis with 1 μL GAD65 191-320+ INS 17-110+ The CKSTHPLSC-pNZ8148 recombinant plasmids were evenly mixed and transferred into a 0.2 cm electroporation cuvette precooled at 4°C; (3.2) Set the electric shock parameters to 2000V, 25μF, 200Ω, and then place the conversion cup in the electric shock tank for electric shock; (3.3) After the electroporation, add 1 mL of GMMC recovery medium, place on ice for 5 min, transfer to a 1.5 mL Enpendof tube, and place in a 30°C incubator for 2 h to recover the culture solution; (3.4) Spread the recovered bacterial solution on GM17 solid medium containing 10 μg / mL chloramphenicol, invert it after the bacterial solution is completely absorbed, and incubate it at 30°C for 36 hours. When transformants appear, pick the positive clones; (3.5) The selected positive clones were inoculated into 5 mL of GM17 broth containing 10 μg / mL chloramphenicol to amplify GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine was mixed with 250μL sterile glycerol + 750μL bacterial solution after positive clone amplification, and then frozen at -40℃ for use.

[0030] Positive clones were identified as follows: A. Pick a clone and place it in 5 mL of GM17 broth containing 10 μg / mL chloramphenicol, and culture it at 30°C overnight.

[0031] B. Use the kit to extract the plasmid and design primers p8148-F:5'-acgcgagcataataaacgg-3'; (SEQ ID NO.9) p8148-R:5'-cgaaagcgaaatcaaacga-3'; (SEQ ID NO.10) Perform PCR verification The PCR reaction system is shown in Table 1.

[0032] Table 1

[0033] The PCR reaction program is shown in Table 2.

[0034] Table 2

[0035] (4) After amplifying the obtained GAD651-585+INS1-110 recombinant lactic acid bacteria vaccine, the steps of using 50ng / mL Nisin to induce expression of the fusion protein are as follows: (4.1) GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine was inoculated at a volume ratio of 1:25 between bacterial solution and GM17 broth containing 10 μg / mL chloramphenicol. The vaccine was inoculated into 200 mL of GM17 broth containing 10 μg / mL chloramphenicol and cultured at 30°C until the bacterial OD 600 is 0.4-0.6, and the culture is obtained; (4.2) Add Nisin to the culture to a final concentration of 50 ng / mL and incubate at 30°C for 4 h to induce expression of the fusion protein; (4.3) Induce the successfully expressed GAD65 191-320+ INS 17-110+ The CKSTHPLSC recombinant lactic acid bacteria vaccine solution (GAD65191-320+INS17-110+CKSTHPLSC recombinant lactic acid bacteria vaccine stock solution) was centrifuged at 4°C and 8000 rpm for 20 min, and the precipitate was collected and diluted to 1×10 with sterile PBS. 10 CFU / mL, stored at -40℃ until use; The GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine titer determination method: Take 100 μL GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine stock solution was diluted with 900 μL PBS. -1 Dilution solution, then draw 100 μL 10 -1 Add 900 μL PBS to dilute to 10 -2 dilution, and so on; 100 μL of dilution from each gradient was spread on GM17 solid medium containing chloramphenicol, and cultured overnight at 30°C under anaerobic conditions to calculate the number of colonies; GAD65 191-320+ INS 17-110+ The titer of CKSTHPLSC recombinant lactic acid bacteria vaccine is equal to "colony count × dilution factor × 10", for example: the dilution factor is 10 8 There are 20 colonies on the plate with a titer of 20×10 8×10=2×10 10 CFU / mL.

[0036] (5) The immunization experiment of the obtained dual-antigen type 1 diabetes vaccine targeting M cells on BALB / c mice is as follows: (5.1) Immunization regimen Six-week-old BALB / c mice were divided into 6 groups, with 10 mice in each group, including None group (normal culture, no treatment required), Blank group (100 μL / mouse PBS intragastrically), NZ9000 group (immunized with NZ9000 lactic acid bacteria, 1×10 10 CFU / mL / mouse), Control group (immunized with NZ9000 lactic acid bacteria containing pNZ8148 plasmid, 1×10 10 CFU / mL / mouse), GI group (immunized GAD65 191-320+ INS 17-110 Lactobacillus vaccine, 1×10 10 CFU / mL / mouse), GIC group (immunized GAD65 191-320+ INS 17-110+ CKSTHPLSC lactic acid bacteria vaccine, 1×10 10 CFU / mL / mouse), and gavage immunization began at the 7th week of age; fasting was carried out 6 hours before immunization. After fasting, each group was gavaged with 100 μL / mouse 6.6% NaHCO3 for 15 minutes to neutralize gastric acid. The initial immunization of each group continued for 7 days, and the same dose and process were used for booster immunization after an interval of 14 days; when BALB / c mice were 13 weeks old, except for the None group, the other groups were modeled with streptozotocin (STZ) after completing the above oral immunization to simulate the pathogenesis of T1D; The method for T1D modeling using STZ is: Two weeks after oral immunization, when BALB / c mice were 13 weeks old, they were intraperitoneally injected with STZ at a dose of 50 mg / kg / mouse for 7 consecutive days to establish a T1D disease model. After the injection cycle, the model was considered successful when a significant decrease in body weight and an increase in blood sugar to 10.3 mmol / L were monitored.

[0037] (5.2) Detection method A. 12 hours after the BALB / c mice completed the immunization procedure, three mice in each group were killed and the intestinal contents were taken. The lactic acid bacteria colonization amount was determined according to the titer determination method described in (4.3); B. After the T1D model of BALB / c mice was established using STZ, tail vein blood was collected at 15 and 21 weeks of age, placed in a 37°C incubator for 1 hour, kept at 4°C overnight, and centrifuged at 250 rpm / min for 15 minutes to obtain serum. Plates were coated with GAD65 and INS peptides, respectively, and the operation was performed according to the instructions of the KPLELISA Kit Anti-Mouse ABTS System kit, with serum dilution ratios of 1:5, 1:10, and 1:20; C. After immunization, BALB / c mice were observed until 26 weeks of age, and their blood glucose was measured from the tail vein every week; meanwhile, their body weight was weighed; D. After 26 weeks of age, BALB / c mice were killed by collecting eye blood; pancreatic tissues were collected for paraffin sectioning for HE staining analysis, and the islet area in the pancreas of each group was counted; spleens of mice were collected and grinded to separate spleen cells; intestinal tissues of mice were collected to extract intestinal lamina propria lymphocytes; cells were stimulated with INS peptide and GAD65 peptide for 48 hours, and anti-mouse CD4-FITC (clone number GK1.5), anti-mouse CD25-APC (clone number PC61.5), and anti-mouse Foxp3-PE (clone number FJK-16s) were added to detect INS and GAD65-specific Treg cells, and cell differentiation results were detected by flow cytometry; E. Using the intestinal lamina propria lymphocytes stimulated as described above, anti-mouse CD80 (clone 16-10A1) and CD11c (clone N418) antibodies were added to detect INS and GAD65-specific DC cells, and the cell differentiation results were detected using a flow cytometer.

[0038] (6) Experimental results The experimental data were analyzed using Graphpad prism8.0.2 statistical software for one-way ANOVA and t-test, with P < 0.05 considered statistically significant.

[0039] (6.1) 12 hours after the BALB / c mice completed the immunization procedure, three mice were killed and the intestinal contents were obtained. The amount of lactic acid bacteria colonization was determined by the titer determination method. The number of lactic acid bacteria colonizing the intestines of BALB / c mice in the GIC recombinant lactic acid bacteria vaccine group was nearly 10 times higher than that in the GI recombinant lactic acid bacteria, Control group, and Blank group. The results are as follows Figure 2 As shown; (6.2) During the experiment, the mice in each group had normal activity, normal body temperature, and granular feces. As of 26 weeks of age, the weight of the Control group was significantly lighter than that of the GI group and the GIC group, and the hyperglycemia in the GIC group was reduced and alleviated compared with the other two groups, proving that the M cell-targeted dual-antigen type 1 diabetes vaccine can more effectively delay or prevent the disease progression of type 1 diabetes than the non-targeted vaccine. The weight and blood sugar results are shown in Figure 3 As shown; (6.3) At the 15th week of age, the oral GIC recombinant lactic acid bacteria vaccine group produced GAD65 and INS specific IgG antibodies with a titer of 1:5; at the 21st week of age, 57% of the mice still maintained the above antibody level. The duration of maintaining antigen release and inducing immune tolerance in the GIC group was significantly higher than that in the GI group. The results are as follows Figure 4 As shown; (6.4) By analyzing the islet area of ​​each group, there was no significant difference in the average area of ​​healthy islets between the GIC group and the None group without any treatment, proving that the GIC vaccine can reduce islet damage during the development of T1D. The results are as follows Figure 5 As shown; (6.5) Flow cytometry analysis revealed that Treg (CD4 + CD25 + Fxop3 + ) cells were significantly increased compared with the GI group and the Control group (P<0.05), indicating that after the autoantigen was expressed by lactic acid bacteria vector and taken orally, under the action of the targeting peptide CKSTHPLSC, the expression of Tregs could be induced in the intestine and spleen. Inducing Treg cell proliferation and differentiation is one of the key factors for using autoantigens to induce the body to rebuild immune tolerance to the antigen. The results of spleen cell Treg flow cytometry are shown in Figure 2. Figure 6 As shown in A, the flow cytometry results of Treg lymphocytes in the intestinal lamina propria are as follows Figure 6 As shown in B; (6.6) Flow cytometry experiments showed that both the GIC and GI groups could induce the proliferation and differentiation of dendritic cells (DCs) secreting interleukin 10 in the intestinal lamina propria lymphocytes, and the effect of the GIC group was more significant. Interleukin 10 is an important cytokine that induces immune tolerance and can inhibit the development of T1D. Figure 6 As shown in C; (6.7) Flow cytometry experiments revealed DCs (CD80 + CD11c + ) cells were significantly inhibited compared with the GI group and the Control group (P<0.05). DC cells are one of the earliest cells found to infiltrate the pancreatic islets during the development of T1D. Inhibiting DC cell proliferation can effectively protect the function of pancreatic β cells and reduce the development of T1D. Compared with the group without expressing the targeting peptide CKSTHPLSC, the dual antigen type 1 diabetes vaccine targeting M cells in the present invention can significantly inhibit the maturation of DC cells, and can well induce immune tolerance against both GAD65 and INS genes, protecting pancreatic islet cells. The results are as follows Figure 6 As shown in D.

[0040] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

[0041] Sequence Listing SEQ ID NO.1

[0042] MGHHHHHHGL AADWLTSTAN TNMFTYEIAP VFVLLEYVTL KKMREIIGWP GGSGDGIFSP 60 GGAISNMYAM MIARFKMFPE VKEKGMAALP RLIAFTSEHS HFSLKKGAAA LGIGTDSVIL 120 IKCDERGKMI PSDLERRILG GGGSWGPDPA AAFVNQHLCG SHLVEALYLV CGERGFFYTP 180 KTRREAEDLQ VGQVELGGGP GAGSLQPLAL EGSLQKRGIV EQCCTSICSL YQLENYCNGG 240 GGSCKSTHPL SC 252 SEQ ID NO.2

[0043] ggtcttgctg ctgattggct tacttcaact gctaacacta acatgttcac ttacgaaatc 60 gctccagttt tcgttcttct tgaatacgtt actcttaaaa aaatgcgtga aatcatcggt 120 tggccaggtg gttcaggtga tggtatcttc tcaccaggtg gtgctatctc aaacatgtac 180 gctatgatga tcgctcgttt caaaatgttc ccagaagtta aagaaaaagg tatggctgct 240 cttccacgtc ttatcgcttt cacttcagaa cactcacact tctcacttaa aaaaggtgct 300 gctgctcttg gtatcggtac tgattcagtt atccttatca aatgtgatga acgtggtaaa 360 atgatcccat cagatcttga acgtcgtatc ctt 393 SEQ ID NO.3

[0044] tggggtccag atccagctgc tgctttcgtt aaccaacacc tttgtggttc acaccttgtt 60 gaagctcttt accttgtttg tggtgaacgt ggtttcttct acactccaaa aactcgtcgt 120 gaagctgaag atcttcaagt tggtcaagtt gaacttggtg gtggtccagg tgctggttca 180 cttcaaccac ttgctcttga aggttcactt caaaaacgtg gtatcgttga acaatgttgt 240 acttcaatct gttcacttta ccaacttgaa aactactgta ac 282 SEQ ID NO.4

[0045] tgtaaatcaa ctcacccact ttcatgt 27 SEQ ID NO.5

[0046] ggtggtggtg gttca 15 SEQ ID NO.6

[0047] catcatcacc atcatcac 18 SEQ ID NO.7

[0048] atggcgagcc cgggcagcgg cttttggagc tttggcagcg aagatggcag cggcgatagc 60 gaaaacccgg gcaccgcgcg cgcgtggtgc caggtggcgc agaaatttac cggcggcatt 120 ggcaacaaac tgtgcgcgct gctgtatggc gatgcggaaa aaccggcgga aagcggcggc 180 agccagccgc cgcgcgcggc ggcgcgcaaa gcggcgtgcg cgtgcgatca gaaaccgtgc 240 agctgcagca aagtggatgt gaactatgcg tttctgcatg cgaccgatct gctgccggcg 300 tgcgatggcg aacgcccgac cctggcgttt ctgcaggatg tgatgaacat tctgctgcag 360 tatgtggtga aaagctttga tcgcagcacc aaagtgattg attttcatta tccgaacgaa 420 ctgctgcagg aatataactg ggaactggcg gatcagccgc agaacctgga agaaattctg 480 atgcattgcc agaccaccct gaaatatgcg attaaaaccg gccatccgcg ctattttaac 540 cagctgagca ccggcctgga tatggtgggc ctggcggcgg attggctgac cagcaccgcg 600 aacaccaaca tgtttaccta tgaaattgcg ccggtgtttg tgctgctgga atatgtgacc 660 ctgaaaaaaa tgcgcgaaat tattggctgg ccgggcggca gcggcgatgg catttttagc 720 ccgggcggcg cgattagcaa catgtatgcg atgatgattg cgcgctttaa aatgtttccg 780 gaaggaaag aaaaaggcat ggcggcgctg ccgcgcctga ttgcgtttac cagcgaacat 840 agccatttta gcctgaaaaa aggcgcggcg gcgctgggca ttggcaccga tagcgtgatt 900 ctgattaaat gcgatgaacg cggcaaaatg attccgagcg atctggaacg ccgcattctg 960 gaagcgaaac agaaggctt tgtgccgttt ctggtgagcg cgaccgcggg caccaccgtg 1020 tatggcgcgt ttgatccgct gctggcggtg gcggatattt gcaaaaaata taaaatttgg 1080 atgcatgtgg atgcggcgtg gggcggcggc ctgctgatga gccgcaaaca taatggaaa 1140 ctgagcggcg tggaacgcgc gaacagcgtg acctggaacc cgcataaaat gatgggcgtg 1200 ccgctgcagt gcagcgcgct gctggtgcgc gaagaggcc tgatgcagaa ctgcaaccag 1260 atgcatgcga gctatctgtt tcagcaggat aaacattatg atctgagcta tgataccggc 1320 gataaagcgc tgcagtgcgg ccgccatgtg gatgtgttta aactgtggct gatgtggcgc 1380 1440 ctgtataaca ttattaaaaa ccgcgaaggc tatgaaatgg tgtttgatgg caaaccgcag 1500 cataccaacg tgtgcttttg gtatattccg ccgagcctgc gcaccctgga agataacgaa 1560 gaacgcatga gccgcctgag caaagtggcg ccggtgatta aagcgcgcat gatggaatat 1620 ggcaccacca tggtgagcta tcagccgctg ggcgataaag tgaacttttt tcgcatggtg 1680 attagcaacc cggcggcgac ccatcaggat attgattttc tgattgaaga aattgaacgc 1740 ctgggccagg atctg 1755 SEQ ID NO.8

[0049] atggccctgt ggatgcgcct cctgcccctg ctggcgctgc tggccctctg gggacctgac 60 ccagccgcag cctttgtgaa ccaacacctg tgcggctcac acctggtgga agctctctac 120 ctagtgtgcg gggaacgagg cttcttctac acacccaaga cccgccggga ggcagaggac 180 ctgcaggtgg ggcaggtgga gctgggcggg ggccctggtg caggcagcct gcagcccttg 240 gccctggagg ggtccctgca gaagcgtggc attgtggaac aatgctgtac cagcatctgc 300 tccctctacc agctggagaa ctactgcaac 330

Claims

1. A method for preparing a dual-antigen type 1 diabetes vaccine targeting M cells, characterized in that: The steps include: Step (1), the 191-320 amino acid sequence of the human glutamic acid decarboxylase 65 gene GAD65, the 17-110 amino acid nucleotide sequence of the insulin INS gene, and the nucleotide sequence of the CKS9 targeting peptide CKSTHPLSC are sequentially connected through the G4S connecting peptide GGGGS, and after the lactic acid bacteria codon preference is optimized, GAD65 191-320 +INS 17-110 +CKSTHPLSC gene fragment was inserted into pNZ8148 vector to construct GAD65 191-320+ INS 17-110+ CKSTHPLSC-pNZ8148 recombinant plasmid; Step (2), GAD65 constructed in step (1) 191-320+ INS 17-110+ The CKSTHPLSC-pNZ8148 recombinant plasmid was transformed into MC1061 competent Escherichia coli for cloning and amplification, and the recombinant plasmid was extracted; Step (3), the recombinant plasmid extracted in step (2) is electroporated into NZ9000 competent Lactococcus lactis to construct GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine; Step (4), GAD65 constructed in step (3) 191-320+ INS 17-110+ After the CKSTHPLSC recombinant lactic acid bacteria vaccine was amplified, Nisin was used to induce the expression of fusion protein.

2. The method for preparing the dual-antigen type 1 diabetes vaccine targeting M cells according to claim 1, characterized in that: In step (1), the nucleotide sequence of amino acids 191-320 of the human glutamate decarboxylase 65 gene GAD65, the nucleotide sequence of amino acids 17-110 of the insulin INS gene, and the nucleotide sequence of the targeting peptide CKSTHPLSC are sequentially connected by a G4S connecting peptide GGGGS. 191-320 +INS 17-110 +CKSTHPLSC nucleotide sequence 5' end with 6HIS tag, after lactic acid bacteria codon preference optimization, the final expression of GAD65 191-320+ INS 17-110+ The amino acid sequence of CKSTHPLSC is shown in SEQ ID NO.1; in: Among them, GAD65 191-320 The nucleotide sequence of the amino acid is shown in SEQ ID NO.2 after optimization of lactic acid bacteria codon preference; INS 17-110 The nucleotide sequence of the amino acid is shown in SEQ ID NO.3 after optimization of lactic acid bacteria codon preference; The nucleotide sequence of the targeting peptide CKSTHPLSC is shown in SEQ ID NO. 4 after optimization of lactic acid bacteria codon preference; The nucleotide sequence of the G4S connecting peptide GGGGS is shown in SEQ ID NO.5 after being optimized for lactic acid bacteria codon preference; The nucleotide sequence of the 6HIS tag is shown in SEQ ID NO.

6.

3. The method for preparing the dual-antigen type 1 diabetes vaccine targeting M cells according to claim 1, characterized in that: In step (1), the method for inserting the pNZ8148 vector is: Located in human GAD65 191-320 The 6His tag nucleotide sequence is directly inserted at the 5' end before the amino acid nucleotide sequence Nco I restriction site, directly inserted at the 3' end of the nucleotide sequence of the targeting peptide CKSTHPLSC XB I restriction site, and then insert Nco I restriction site and XB I restriction site nucleotide sequence and pNZ8148 vector, using Nco I enzyme, XB I enzyme double digestion, followed by T4 enzyme ligation overnight to construct the recombinant plasmid GAD65 191-320+ INS 17-110+ CKSTHPLSC-pNZ8148.

4. The method for preparing the dual-antigen type 1 diabetes vaccine targeting M cells according to claim 1, characterized in that: The specific method of step (2) is: (2.1) GAD65 191-320+ INS 17-110+ The CKSTHPLSC-pNZ8148 recombinant plasmid was transformed into MC1061 competent Escherichia coli, spread on LB solid medium plates containing chloramphenicol, and cultured inverted at 37°C overnight. Single colonies on the LB solid medium plates containing chloramphenicol were picked for positive clone identification; (2.2) Add the positive clones to a test tube containing LB liquid culture medium containing chloramphenicol and culture them in a constant temperature shaker at 37°C overnight. The next day, inoculate the bacterial liquid into LB liquid culture medium containing chloramphenicol and culture them in a constant temperature shaker at 37°C overnight. The next day, use a plasmid extraction kit to extract GAD65 from the bacterial liquid harvested from the clone amplification. 191-320+ INS 17-110+ CKSTHPLSC-pNZ8148 recombinant plasmid.

5. The method for preparing the dual-antigen type 1 diabetes vaccine targeting M cells according to claim 4, characterized in that: The constant temperature shaker speed is 200 rpm / min.

6. The method for preparing the dual-antigen type 1 diabetes vaccine targeting M cells according to claim 1, characterized in that: The specific method of step (3) is: (3.1) Mix 40 μL NZ9000 competent Lactococcus lactis with 1 μL GAD65 191-320+ INS 17-110+ The CKSTHPLSC-pNZ8148 recombinant plasmids were evenly mixed and transferred into a 0.2 cm electroporation cuvette precooled at 4°C; (3.2) Set the electric shock parameters to 2000V, 25μF, 200Ω, and then place the conversion cup in the electric shock tank for electric shock; (3.3) After the electroporation, add 1 mL of GMMC recovery medium, place on ice for 5 min, transfer to a 1.5 mL Enpendof tube, and place in a 30°C incubator for 2 h to recover the culture solution; (3.4) Spread the recovered bacterial solution on GM17 solid medium containing 10 μg / mL chloramphenicol, invert it after the bacterial solution is completely absorbed, and incubate it at 30°C for 36 hours. When transformants appear, pick the positive clones; (3.5) The selected positive clones were inoculated into 5 mL of GM17 broth containing 10 μg / mL chloramphenicol to amplify GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine.

7. The method for preparing the dual-antigen type 1 diabetes vaccine targeting M cells according to claim 1, characterized in that: In step (4), the specific method of inducing the expression of the fusion protein with Nisin is: (4.1) GAD65 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine was inoculated at a volume ratio of 1:25 between bacterial solution and GM17 broth containing 10 μg / mL chloramphenicol and cultured at 30°C until the bacterial OD 600 is 0.4-0.6, and the culture is obtained; (4.2) Add Nisin to the culture to a final concentration of 50 ng / mL and incubate at 30°C for 4 h to induce expression of the fusion protein; (4.3) Induce the successfully expressed GAD65 191-320+ INS 17-110+ The CKSTHPLSC recombinant lactic acid bacteria vaccine solution was centrifuged at 4°C, the precipitate was collected and diluted to 1×10 10 CFU / mL and stored at -40℃ until use.

8. A dual-antigen type 1 diabetes vaccine targeting M cells prepared by the method for preparing a dual-antigen type 1 diabetes vaccine targeting M cells according to any one of claims 1 to 7.

9. GAD65 obtained by the method for preparing a dual-antigen type 1 diabetes vaccine targeting M cells according to any one of claims 1 to 7 191-320+ INS 17-110+ CKSTHPLSC recombinant lactic acid bacteria vaccine.

10. GAD65 according to claim 9 191-320+ INS 17-110+ Application of CKSTHPLSC recombinant lactic acid bacteria vaccine in the preparation of dual-antigen type 1 diabetes vaccine targeting M cells.

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