CN112773889A

CN112773889A - VDBP/VSIG 4-based immune negative regulation multivalent vaccine and preparation method thereof

Info

Publication number: CN112773889A
Application number: CN202011479150.7A
Authority: CN
Inventors: 杨曌
Original assignee: Yongchuan Hospital of Chongqing Medical University
Current assignee: Yongchuan Hospital of Chongqing Medical University
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2021-05-11

Abstract

The invention discloses an immune negative regulation multivalent vaccine based on VDBP/VSIG4 and a preparation method thereof, wherein the vaccine is a compound of a VDBP multivalent epitope peptide and a VSIG4 gene recombinant expression vector; the VDBP1 multivalent epitope peptide/VDBP 2 multivalent epitope peptide respectively consists of epitopes VDBP 211-219/VDBP 235-243, a transmembrane sequence LWMRWYSPK and a linker sequence, wherein the transmembrane sequence is positioned at an amino end, the epitopes VDBP 211-219/VDBP 235-243 are positioned at a carboxyl end, and the epitopes are connected with the transmembrane sequence by the linker sequence; the vaccine can enter cells through a transmembrane sequence, effectively promotes the type 1 diabetes epitope VDBP1/VDBP2 to enter an antigen presentation way, further synthesizes VSIG4 protein, inhibits the response of VDBP specific CD8+ T cells, achieves the aim of reducing immune response, and thus prolongs the course of disease of type 1 diabetes.

Description

VDBP/VSIG 4-based immune negative regulation multivalent vaccine and preparation method thereof

Technical Field

The invention relates to a multivalent vaccine, in particular to a double-target immune negative regulation multivalent vaccine for type 1 diabetes, and also relates to a preparation method of the vaccine.

Background

The prevalence of diabetes has increased year by year in the world in recent years, and recent epidemiological investigations have shown that the total number of diabetes patients is expected to reach 6.42 billion in the world by 2040 years. Type 1 and type 2 diabetes are common in diabetes typing, wherein type 1 diabetes (type 1 dib T1DM) accounts for 10-15% of the total number of diabetes, and is an autoimmune disease which is mediated by T lymphocytes, has islet beta cell injury and islet function impairment, and causes absolute deficiency of insulin secretion. The pathogenesis of T1DM includes various factors such as heredity, diet and environment.

Vitamin D Binding Protein (VDBP) is globulin, is a 58k Da glycoprotein, is used as a main carrier protein of plasma vitamin D and metabolites thereof, supports the bioavailability of active 1, 25-dihydroxyvitamin D [1,25(OH)2D ] and a precursor thereof, namely 25-hydroxyvitamin D [25(OH) D ], and has the most important function of maintaining the bioactivity of the vitamin D. VDBP is reabsorbed by macrophage-mediated endocytosis as a 25- (OH) VitD3/VDBP complex and is catabolized by proximal tubule epithelial cells, helping to reduce its urinary excretion level. Recent studies have shown that patients with type 1 diabetes (T1DM) or type 2 diabetes (T2DM) have increased urinary VDBP with normal proteinuria, microalbuminuria, and macroproteinuria. Therefore, VDBP is expected to be a relevant antigen for type 1 diabetes.

A newly identified B7 family-related protein VSIG4(V-set and immunoglobulin domain-binding 4), also known as Ig superfamily protein-39 (Ig superfamily protein 39, Z39 Ig). VSIG4 is specifically expressed on tissue macrophages such as peritoneal macrophages (PEMs), liver Kupffer cells. Initial studies have shown that VSIG4 mediates clearance of C3 b-conditioned pathogens by binding to its ligand C3b/i C3b as a complement receptor. VSIG4+ macrophages were found to be associated with diabetes resistance in a mouse model of type i diabetes (non-obese). Also, VSIG4 binds to unknown ligands on T cells as orphan ligands, inhibiting T cell proliferation, activation, and IL-2 production. Suggesting that VSIG4 may transmit anti-inflammatory signals in inflammatory pathological lesions.

Due to the barrier effect of the cell membrane, biological macromolecules cannot freely enter the cell. In recent years, small-molecule polypeptides with cell membrane penetrating capacity are discovered successively, can effectively carry exogenous macromolecules into cells, and have no obvious toxic or side effect on host cells. These polypeptides having cell-penetrating ability are named cell-penetrating peptides (CPPs). LWMRWYSPK has been found to be effective in promoting foreign epitopes conjugated thereto into the MHC-I antigen presentation pathway, with significantly enhanced kinetics, and with proteasome/TAP independent and aminopeptidase dependent processes.

Disclosure of Invention

In view of the above, the present invention provides an immunomodulatory multivalent vaccine for type 1 diabetes mellitus, and provides a method for preparing the immunomodulatory multivalent vaccine for type 1 diabetes mellitus.

In order to achieve the purpose, the invention adopts the following technical scheme:

1. an immunoregulatory multivalent vaccine for type 1 diabetes characterized by: the vaccine is a compound of VDBP1 multivalent epitope peptide, VDBP2 multivalent epitope peptide and VSIG4 gene recombination expression vector;

the VDBP1 multivalent epitope peptide consists of a transmembrane sequence LWMRWYSPK, a linker sequence: SYSY, diabetes-related antigen VDBP protein CD8 cell epitope VDBP 211-219: LLTTLSNRV;

the VDBP2 multivalent epitope peptide consists of a junction sequence LWMRWYSPK: SYSY, diabetes-related antigen VDBP protein CD8 cell epitope VDBP 235-243: NLIKLAQKV;

in the VDBP1 epitope peptide and the VDBP2 epitope peptide, the transmembrane sequence is positioned at an amino terminal, the CD8 cell epitope sequence is positioned at a carboxyl terminal, and the epitope and the transmembrane sequence or the epitope are connected by a linker sequence.

2. The immuno-negatively regulated multivalent vaccine for type 1 diabetes mellitus according to claim 1, characterized in that: the linker sequence is SYSY.

3. The immuno-negatively regulated multivalent vaccine for type 1 diabetes mellitus according to claim 1, characterized in that: the molar ratio of the VDBP1 epitope peptide to the VDBP2 epitope peptide is 1: 1.

4. The immuno-negatively regulated multivalent vaccine for type 1 diabetes mellitus according to claim 1, characterized in that: the VSIG4 gene recombinant expression vector is obtained by inserting a VSIG4 sequence into a eukaryotic expression vector pAZV 5Z.

5. A method of preparing an immuno-negatively regulated multivalent vaccine for type 1 diabetes mellitus as claimed in claim 1, wherein: adding 0.5% KHCO dissolved with VSIG4 gene recombinant expression vector under stirring₃VDBP1 epitope peptide and V are dropwise dissolved in the solutionAnd (3) continuously stirring the aqueous solution of the DBP2 epitope peptide for 20 minutes after the dripping is finished, and standing for 20 minutes to obtain the DBP2 epitope peptide.

6. The method of preparing an immuno-negatively regulated multivalent vaccine for type 1 diabetes according to claim 5, wherein: the molar ratio of the VDBP1 epitope peptide to the VDBP2 epitope peptide is 1: 1.

The invention has the beneficial effects that: the vaccine can enter cells through a transmembrane sequence, effectively promotes the type 1 diabetes epitope VDBP1/VDBP2 to enter an antigen presentation way to excite specific CD8+ T cell response, simultaneously VSIG4 gene is expressed in the cells to generate VSIG4 protein, induces specific CD8+ T cell immunosuppression, achieves the aim of reducing immune response, and thus prolongs the course of disease of type 1 diabetes.

Animal experiment results prove that the vaccine can inhibit proliferation of specific CD8+ T cells and secretion of cytokines, further reduce the morbidity of diabetic mice, relieve damage of islet cells and achieve the effect of treating type 1 diabetes. The vaccine provided by the invention is simple in preparation method and low in cost, and has a good development and application prospect in the field of type 1 diabetes prevention.

Drawings

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings, in which:

FIG. 1 is a schematic diagram of the primary structures of VDBP1 epitope peptide and VDBP2 epitope peptide;

FIG. 2 is an agarose gel electrophoresis identification of the gene VSIG 4;

FIG. 3 is a diagram showing the electrophoretic identification of the product of double restriction enzymes of the recombinant eukaryotic expression plasmid of VSIG 4;

FIG. 4 is a transmission electron micrograph of a vaccine of the present invention;

FIG. 5 shows the result of immunoblotting (Western Blot) of VDBP/VSIG of the multivalent vaccine;

FIG. 6 shows the results of measurement of the proliferative capacity of lymphocytes;

FIG. 7 shows the results of measurement of cytokine secretion ability of lymphocytes;

FIG. 8 is the mean incidence of diabetes in NOD mice;

FIG. 9 shows the islet mean survival detection node

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified, in the preferred examples are generally carried out according to conventional conditions, for example, as described in the molecular cloning protocols (third edition, J. SammBruk et al, Huangpetang et al, scientific Press, 2002), or according to the conditions recommended by the manufacturers.

Preparation of immune negative regulation multivalent vaccine for type I and type 1 diabetes

1. Design and synthesis of VDBP1 epitope peptide and VDBP2 epitope peptide

The primary structures of the VDBP1 epitope peptide and the VDBP2 epitope peptide designed by the invention are shown in figure 1, and each epitope is connected with a transmembrane sequence or an epitope peptide sequence by a linker sequence, so that each epitope can keep independence and can be effectively presented. In this example, the linker sequence is serine-tyrosine-serine-tyrosine (Ser-Tyr-Ser-Tyr, SYSY); the amino acid sequence of the VDBP1 epitope peptide is shown as SEQ ID No.1, and the amino acid sequence of the VDBP2 epitope peptide is shown as SEQ ID No. 2; meanwhile, an Ovalbumin (OVA) epitope peptide is used as a contrast peptide (consisting of an epitope OVA257-264, a transmembrane sequence HIV-Tat49-57, an endoplasmic reticulum retention signal sequence KDEL and a linker sequence, and the amino acid sequence of the epitope peptide is shown as SEQ ID No. 3.

The synthesis of the polypeptide was carried out on an ABI 431A solid phase polypeptide synthesizer (PE company, USA). The method employed a standard fluorenylmethyloxycarbonyl (Fmoc) protocol with arginine using two couplings. 0.125mmol of p-methylol phenoxymethyl polystyrene resin (HMP resin) is selected initially, and peptide chains are extended from carboxyl terminal to amino terminal one by one according to a polypeptide sequence, wherein the dosage of each amino acid is 0.5mmol, and the molar ratio of each amino acid to the resin is 4: 1. The alpha-amino group of each amino acid is protected by Fmoc, and the protecting groups of the other side chains are respectively as follows: lys (Boc), Ser (tBu), Glu (OtBu), Arg (Pmc), His (Trt), Thr (tBu), and Tyr (tBu). The first amino acid was attached to the resin using 4-Dimethylaminopyridine (DMAP), the amino acid was activated using 1-hydroxybenzotriazole (HOBt) and Dicyclohexylcarbodiimide (DCC), and after coupling the Fmoc protecting group was removed using 20% volume fraction piperidine in water. After polypeptide synthesis, the resin-crude peptide product is mixed in 10mL of cutting fluid A (composed of 0.75g of crystallized phenol, 0.25mL of 1, 2-Ethanedithiol (EDT), 0.5mL of thioanisole, 0.25mL of deionized water and 10mL of trifluoroacetic acid (TFA) under an ice bath condition), and after the temperature of the cutting fluid to be cut rises to room temperature, the reaction is carried out for 2 hours under stirring, so that a peptide chain is cleaved from the resin, and meanwhile, various protecting groups are removed. The reaction mixture was filtered through a G4 glass frit funnel to remove the resin, and the reaction flask, resin and funnel were repeatedly rinsed with 1mL TFA followed by 5-10 mL dichloromethane. Evaporating the filtrate to 1-2 mL at normal temperature and low pressure, adding 50mL of precooled ether to precipitate the polypeptide, standing overnight at4 ℃, filtering by a G6 glass sand funnel, and vacuumizing to obtain a crude polypeptide product, and storing at-20 ℃ for later use.

The crude polypeptide was dissolved in dimethyl sulfoxide (DMSO) to prepare a 20mg/mL solution, which was filtered through a 0.45 μm pore size microporous membrane and purified by SOURCE gel column chromatography on an AKTA explorer 100 medium pressure liquid chromatograph (Amersham bioscience, Sweden). The mobile phase A consists of 10 volume percent of ethanol and 0.1 volume percent of TFA, and the mobile phase B consists of 90 volume percent of ethanol and 0.1 volume percent of TFA; the elution gradient was: eluting with 1.5 column volumes of mobile phase A, eluting with a mixture of mobile phase A and mobile phase B (the volume fraction of mobile phase B in the mixture gradually increases from 0% to 80% in 8 column volumes), eluting with a mixture of mobile phase A and mobile phase B (the volume fraction of mobile phase B in the mixture gradually increases from 80% to 100% in 0.5 column volumes), collecting polypeptide solution at main peak, freeze drying to obtain pure polypeptide, dissolving with DMSO, and storing at-20 deg.C for use.

The purity of the pure polypeptide is determined by a Delta 600 high pressure liquid chromatograph (Waters company, USA), a Symmetry Shield C18 column is adopted, a mobile phase consists of acetonitrile with the volume percentage of 10-60% and TFA with the volume percentage of 0.1%, and the mobile phase is eluted in a gradient way with the flow rate of 1 mL/min. The results show that the purity of the synthesized polypeptide reaches more than 90 percent. Meanwhile, the pure polypeptide product is used for measuring the molecular weight by an API 2000LC/MS type electrospray ionization mass spectrometer. The results show that the molecular weights of the synthesized polypeptides all agree with theoretical values.

2. Construction of recombinant expression vector of VSIG4 Gene

(1) Cloning of the full-Length coding Gene of VSIG4

Designing and synthesizing upstream and downstream primers according to the VSIG4 gene sequence with GenBank accession number NC-000086.8, wherein the sequences are shown as SEQ ID No.4 and SEQ ID No. 5. PCR primers to amplify the VSIG4 full-length coding gene; carrying out PCR by taking a human placenta cDNA library as a template, wherein the PCR conditions are as follows: pre-denaturation at 94 ℃ for 3 min, then denaturation at 94 ℃ for 30 sec, annealing at 56 ℃ for 30 sec, extension at 72 ℃ for 30 sec for 30 cycles, and finally extension at 72 ℃ for 5 min; after agarose gel electrophoresis identification and gel recovery kit gel cutting recovery purification, the PCR product is connected with a vector pGEM-T, the connecting product transforms escherichia coli JM109 competent cells, a culture medium containing Amp/IPTG/X-GAL is used for blue-white spot screening, white spot culture is selected, plasmids are extracted, Shanghai's pharmaceutical company is entrusted to determine gene sequences, and positive clone plasmids with correct sequences are named as pGEM-T/VSIG 4;

the agarose gel electrophoresis identification pattern of the PCR product is shown in FIG. 2, wherein lane M is a DNA molecular weight standard, lane 1 is a PCR product, and lane 1 shows a single specific band at about 1200bp, which is consistent with the expected result; sequencing of the plasmid showed that the inserted gene sequence was identical to the full-length gene encoding VSIG 4.

(2) Preparation of VSIG4 gene recombinant eukaryotic expression vector

Designing and synthesizing a PCR primer according to the full-length coding gene sequence of the VSIG4 and the multiple cloning site of the eukaryotic expression vector pAZV5Z, wherein the sequences are shown as SEQ ID No.6 and SEQ ID No. 7; amplifying a cDNA fragment containing a full-length coding gene of VSIG4, an XBI enzyme cutting site at the 5 'end and an Ecor1 enzyme cutting site at the 3' end; carrying out PCR by taking pGEM-T/VSIG4 as a template, wherein the PCR condition is the same as that in the step (1); carrying out agarose gel electrophoresis identification on a PCR product, cutting gel by a gel recovery kit, recovering and purifying, carrying out double enzyme digestion by using restriction enzymes XBI and Ecor1, carrying out double enzyme digestion on a double enzyme digestion product by the gel recovery kit, recovering and purifying the double enzyme digestion product by using the gel recovery kit, connecting the double enzyme digestion product with pAZV5Z which is also subjected to double enzyme digestion by XBI and Ecor1 under the action of T4 DNA ligase, transforming a escherichia coli TOP10 competent cell by using the connection product, carrying out blue-white spot screening by using a culture medium containing Amp/IPTG/X-GAL, picking out white spot culture, extracting plasmids, carrying out double enzyme digestion identification by using XBI and Ecor1, entrusting a Shanghai's pharmaceutical company to determine a gene sequence, and naming a positive clone with a correct sequence and a reading frame as pAZV5Z/VSIG 4;

the agarose gel electrophoresis identification picture of the recombinant eukaryotic expression plasmid double enzyme digestion product is shown in figure 3, wherein a lane M is a DNA molecular weight standard, a lane 1 is a double enzyme digestion product, and two electrophoresis bands appear in the lane 1 at about 3000bp and 1000bp, which is consistent with the expected result; the sequencing result of the plasmid shows that the inserted gene sequence has no mutation, the reading frame is correct, and no frame shift exists.

3. Preparation of compound of VDBP1 epitope peptide, VDBP2 epitope peptide and VSIG4 gene recombinant expression vector

Subjecting pAZV5Z/VSIG4 to KHCO concentration of 0.5%₃Dissolving to prepare a solution with the concentration of 500 mug/mL, taking 100 mug L of the solution, dripping 100 mug L of polypeptide solution (consisting of VDBP1 epitope peptide and VDBP2 epitope peptide with the molar ratio of 1: 1) with the total concentration of 1000 mug/mL at the speed of 5 mug/min under the condition of mixed rotation, continuing the mixed rotation for 20 minutes after finishing the dripping, and standing for 20 minutes to obtain a compound of the VDBP1 epitope peptide, the VDBP2 epitope peptide and a VSIG4 gene recombinant expression vector, namely the immune negative regulation multivalent vaccine for the type 1 diabetes.

Transmission electron microscopy analysis: dropping the new-prepared compound on a 200-mesh copper net, adsorbing for 3 minutes, blotting the new-prepared compound by using absorbent paper, airing for 30 seconds, carrying out negative dyeing on the new-prepared compound by using a 1% by mass volume aqueous solution of uranium acetate for 30 seconds, blotting the new-prepared compound by using the absorbent paper, airing for 30 seconds, and observing by using a 80kV transmission electron microscope. The results are shown in FIG. 4, where the resulting composites are in the form of nearly round particles of uniform size, with the majority of the particles having a major dimension of less than 25 nm.

Preparation of two-multivalent vaccine VDBP/VSIG4 transfection dendritic cell vaccine

1. Sorting of dendritic cells

Taking 4-week-old Balb/c mice, removing neck, killing, soaking in 75% ethanol solution for 5 min, cutting skin and subcutaneous tissue under aseptic condition, and separating miceWashing femur and tibia with 0.01mol/L sterile PBS 3-5 times, cutting off two ends of femur and tibia, repeatedly flushing medullary cavity with 0.01mol/L sterile PBS to obtain single cell suspension, centrifuging at 1000r/min for 5 min to remove fat layer, resuspending cells with 0.01mol/L sterile PBS, slowly adding equal volume Percoll cell separating medium, centrifuging at 3000r/min for 30 min, collecting mononuclear cells, washing with 0.01mol/L sterile PBS for 2 times, resuspending with serum-containing DMEM culture medium, inoculating the obtained single cell suspension into T-25 culture flask, and supplementing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4(IL-4) at 37 deg.C and CO₂Culturing in an incubator with gas volume fraction of 5% and saturated humidity, supplementing cell factors with half of every other day of liquid change, and harvesting cells on day 7 to obtain dendritic cells.

2. Multivalent vaccine VDBP/VSIG4 transfected dendritic cells

Dendritic cells were cultured at a cell concentration of 1X 10⁶Inoculating each cell/ml in a 6-well plate, adding 10 mu g of multivalent vaccine VDBP/VSIG4, collecting cells 48 hours after infection, washing with PBS and resuspending to obtain the VDBP/VSIG4 virus transfected dendritic cells.

Western Blot detection: carrying out ultrasonic lysis on VDBP/VSIG4 transfected dendritic cells and empty plasmid transfected dendritic cells respectively, collecting total cell protein, carrying out SDS-PAGE, carrying out electrophoresis, then carrying out electrotransfer on a PVDF membrane, washing the membrane, sealing, adding a rabbit anti-human VSIG4 polyclonal antibody, incubating for 1 hour at 37 ℃, washing the membrane, adding goat anti-rabbit IgG, incubating for 1 hour at 37 ℃, washing the membrane, and developing. The results are shown in FIG. 3, where lane 1 is: PBS, lane 2: empty plasmid multivalent vaccine, lane 3 is: multivalent vaccine VDBP/VSIG 4. The results show that: VDBP/VSIG4 can express VSIG4 in dendritic cells.

Detection of anti-type 1 diabetes capacity of multivalent vaccine VDBP/VSIG4 transfection dendritic cell vaccine

2-month-old diabetic-susceptible NOD mice were randomly assigned to two groups: experiment group and control group, experiment group tail vein back transfusion multivalent vaccine VDBP/VSIG4 transfection dendritic cell vaccine, continuous back transfusion 3 times, 1 time per week, each time input 1 × 10⁶(ii) individual cells; normal feeding of control group。

1. Lymphocyte proliferation potency assay

1 week after the last reinfusion, two groups of mice are killed by breaking neck, spleens of the mice are taken under aseptic condition, ground into cell suspension by a 100-mesh screen, separated by a conventional ficoll-diatrizoate stratified fluid gradient centrifugation method to obtain splenic lymphocytes, and the cell concentration is adjusted to 1 × 10 by using RPMI 1640 medium containing 10% fetal calf serum in volume fraction⁶Inoculating into 96-well plate with 100 μ l per well, setting 3 multiple wells per group, adding VSIG4 recombinant protein to final concentration of 1mg/ml per well, standing at 37 deg.C and CO₂Culturing in an incubator with gas volume fraction of 5% for 96 hr, and adding 0.1ml of 1 × 10³Mu Ci/L3H-thymidine (3H-TdR) solution, continuously culturing for 12 hours, rinsing the cells with PBS 3 times, fixing with formaldehyde for 10 minutes, rinsing with trichloroacetic acid solution with volume fraction of 5% pre-cooled at4 ℃ for 3 times, adding 0.5ml NaOH solution with concentration of 0.3mol/L into each hole, carrying out water bath reaction at 60 ℃ for 30 minutes, cooling to room temperature, transferring into a scintillation flask, adding 5ml scintillation fluid, counting the scintillation times (cpm) per minute by using a liquid scintillation counter, measuring the DPM value (reflecting the cell DNA synthesis rate), and repeatedly measuring for 3 times.

The results are shown in FIG. 4, the cpm value of the mouse lymphocyte in the experimental group is 36000 +/-3200, while the cpm value of the mouse lymphocyte in the control group is 55000 +/-6300, and the VDBP/VSIG4 virus-transfected dendritic cell vaccine is proved to be capable of effectively reducing the proliferation capacity of the lymphocyte.

2. Detection of cytokine secretion ability of lymphocytes

1 week after the last reinfusion, two groups of mice are killed by breaking neck, spleens of the mice are taken under aseptic condition, ground into cell suspension by a 100-mesh screen, separated by a conventional ficoll-diatrizoate stratified fluid gradient centrifugation method to obtain splenic lymphocytes, and the cell concentration is adjusted to 1 × 10 by using RPMI 1640 medium containing 10% fetal calf serum in volume fraction⁶Inoculating to 96-well plate (100 μ l per well), setting 3 multiple wells per group, adding GAD65 recombinant protein to final concentration of 1mg/ml per well, standing at 37 deg.C and CO₂Culturing in an incubator with gas volume fraction of 5% for 96 hr, and detecting by ELISA methodInterleukin 2(IL-2) and interferon-gamma (IFN-gamma).

As a result, as shown in FIG. 5, the IL-2 and IFN-y contents of the lymphocytes of the experimental group of mice were 53.5. + -. 5.9pg/ml and 48.4. + -. 5.3pg/ml, respectively, while the IL-2 and IFN-y contents of the lymphocytes of the control group of mice were 126.8. + -. 15.3pg/ml and 102.7. + -. 11.4pg/ml, respectively, confirming that the VDBP/VSIG4 virus-transfected dendritic cell vaccine was effective in reducing the cytokine-secreting ability of the lymphocytes.

3. NOD mouse diabetes incidence detection

Blood glucose levels were monitored in the experimental and control mice.

The results are shown in FIG. 6, the experimental group mice begin to have blood sugar abnormality at 18 weeks of age, the incidence rate of diabetes at 30 weeks of age is 30%, while the control group mice begin to have blood sugar abnormality at 14 weeks of age, and the incidence rate of diabetes at 30 weeks of age is up to 70%; the VDBP/VSIG4 virus-transfected dendritic cell vaccine is proved to be capable of effectively delaying the onset time of diabetes of NOD mice and reducing the incidence rate of the diabetes.

4. Transplanted islet survival detection

Islet cell isolation and purification: killing a C57BL/6J mouse at a dislocated cervical vertebra, soaking and sterilizing the killed mouse by using an ethanol solution with the volume fraction of 75%, opening an abdominal cavity under an aseptic condition, exposing a common bile duct, ligating an opening of a cholepancreatic duct in duodenum under a dissecting microscope, reversely infusing Hank's solution (containing collagenase P with the concentration of 0.1g/L and DNase-I with the concentration of 10 mg/L) along the common bile duct, immediately removing pancreatic tissues after the pancreas expands, cleaning the pancreas tissues in Hank's solution precooled at the temperature of 4 ℃ for 2 times, and shearing the pancreas tissues into 0.5-1 mm³The small blocks are subjected to oscillatory digestion at 37 ℃ for 10-15 minutes until the small blocks are flocculent, Hank's solution precooled at4 ℃ is immediately added to stop digestion, the small blocks are washed for 2 times by the Hank's solution, cell precipitates are resuspended by the Hank's solution, Histopaque with the mass fraction of 84%, 67% and 50% is sequentially added to the cell precipitates, 1500g is centrifuged for 10 minutes, 84-67% and 67-50% layers of cell precipitates are absorbed, the small blocks are washed for 2 times by the Hank's solution, a DMEM culture medium (containing 5.6mmol/L glucose, 10% fetal calf serum, 10U/L penicillin and 100mg/L streptomycin) is used for resuspension, and sampling is carried out to count the pancreatic islet cells。

Islet cell transplantation: 5X 10 mice were tested 1 week after the abnormal blood glucose was detected in NOD mice in the experimental group and the control group, respectively⁵Islet cells from individual C57BL/6J mice were transplanted into the peritoneum of NOD mice, and blood glucose was measured daily, and when blood glucose levels returned to 11.1mM, the transplantation was considered successful, and the survival of two groups of transplanted islets was examined.

As shown in FIG. 7, the mean survival time of transplanted islets of Langerhans of experimental mice was 10 days, while that of transplanted islets of control mice was 4 days, which confirmed that the VDBP/VSIG4 virus-transfected dendritic cell vaccine was effective in prolonging the survival time of transplanted islets of NOD mice.

Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

<110> Chongqing medical university's affiliated Yongchuan Hospital

<120> VDBP/VSIG 4-based immune negative regulation multivalent vaccine and preparation method thereof

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Claims

1.1 an immunomodulatory multivalent vaccine for diabetes mellitus, characterized by: the vaccine is a compound of VDBP1 multivalent epitope peptide, VDBP2 multivalent epitope peptide and VSIG4 gene recombination expression vector;

3. The immuno-negatively regulated multivalent vaccine for type 1 diabetes mellitus according to claim 1, characterized in that: the molar ratio of the VDBP1 epitope peptide to the VDBP2 epitope peptide is 1:1: 1.

5. A method of preparing an immuno-negatively regulated multivalent vaccine for type 1 diabetes mellitus as claimed in claim 1, wherein: adding 0.5% KHCO dissolved with VSIG4 gene recombinant expression vector under stirring₃Dropwise adding an aqueous solution for dissolving VDBP1 epitope peptide and VDBP2 epitope peptide into the solution, continuously stirring for 20 minutes after dropwise adding is finished, and standing for 20 minutes to obtain the polypeptide.