DNA VACCINE CAPABLE OF EFFECTIVELY TREATING AND/OR PREVENTING TYPE 1 DIABETES AND USE THEREOF
20220362359 · 2022-11-17
Assignee
Inventors
Cpc classification
A61K2039/6037
HUMAN NECESSITIES
International classification
Abstract
Provided is use of a recombinant nucleic acid construct containing a B7-2-PE40 exotoxin fusion gene in the preparation of a DNA vaccine or medicament for treatment and/or prevention of type 1 diabetes. The DNA vaccine can reduce blood glucose in patient with type 1 diabetes, restore the secretion of insulin of the patients per se, and reduce the contents of islet cell autoantibody (ICA) and glutamate decarboxylase autoantibody (GAD) in the patients.
Claims
1.-10. (canceled)
11. A method for preventing and/or treating type 1 diabetes in a subject, the method comprising administering to a subject in need thereof an effective amount of (a) a recombinant nucleic acid construct comprising a B7-2-PE40 exotoxin fusion gene or (b) a DNA vaccine or a pharmaceutical composition comprising said recombinant nucleic acid construct.
12. The method according to claim 11, wherein the B7-2-PE40 exotoxin fusion gene is operably ligated to a recombinant expression vector, and the recombinant expression vector is selected from the group consisting of pcDNA3.1/Zeo(+), pVAX1, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, pSVL and adenovirus vector.
13. The method according to claim 12, the recombinant expression vector is pcDNA3.1/Zeo(+).
14. The method according to claim 11, wherein the B7-2-PE40 exotoxin fusion gene has a sequence as shown in SEQ ID NO:1.
15. The method according to claim 11, wherein the DNA vaccine further comprises a pharmaceutically acceptable immune adjuvant.
16. The method according to claim 11, wherein the subject is immunized with the recombinant nucleic acid construct or the DNA vaccine by injection, mucosa, or gene gun introduction.
17. The method according to claim 11, wherein the subject is immunized with the recombinant nucleic acid construct or the DNA vaccine by a manner selected from the group consisting of intravenous injection, intra-arterial injection, intramuscular injection, subcutaneous injection, organ injection, intrathoracic injection and intraperitoneal injection.
18. The method according to claim 11, wherein the DNA vaccine is an aqueous solution or a lyophilized powder for reconstitution for injection or mucosal administration.
19. The method of claim 11, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
20. The method of claim 11, wherein the subject is a mammal.
21. The method of claim 11, wherein the subject is a human.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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SPECIFIC MODELS FOR CARRYING OUT THE INVENTION
[0051] The embodiments of the present invention are described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, they are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.
[0052] In the examples, the expression vectors pcDNA3.1/Zeo(+), Zeocin™, Trizol and Lipofectamine™2000 were purchased from Invitrogen Company. Jurkat and Raji cell lines were purchased from ATCC (American Type Culture Collection). CHO-K1-RPE.40 cell line (Chinese hamster ovary cell anti-Pseudomonas aeruginosa exotoxin cell line) was also purchased from ATCC of the United States; PEA polyclonal antibody, CD86 monoclonal antibody and TMB substrate chromogenic solution were purchased from Sigma Company. PVDF membrane and Amicon Ultra-4 were purchased from Millipore Company. ECL was purchased from Pierce Corporation. MTS (CellTiter 96AQueous One Solution Cell Proliferation Assay) and PureYieild™ plasmid midiprep system were purchased from Promega. KpnI enzyme, XbaI enzyme and T4 ligase were purchased from TaKaRa Company. 2×Pfu PCR MasterMix was purchased from TIANGEN Company. QIAquick Gel Extraction Kit was purchased from QIAGEN Company.
[0053] The main instruments used in the examples were: 9700 PCR instrument (PerkinElmer), Du®640 UV detector (Beckman), Mini II protein electrophoresis instrument and protein semi-dry electrophoresis instrument (Bio-Rad), Gel-Pro3.1 Gel-Imaging System (Media Cybermetic).
Example 1. Establishment of NOD/LTJ Mouse Model of Type 1 Diabetes
[0054] Experimental group: The internationally recognized spontaneous type 1 diabetes mouse model NOD/LTJ (purchased from the Institute of Laboratory Animals, Chinese Academy of Medical Sciences) was selected and prepared according to conventional methods. Female NOD/LTJ mice, aged 7-8 week, were selected. From the 8.sup.th week of age, trace blood in the tail tip of the mice was measured every week, and the appearance of diabetes symptoms was observed. Generally, the disease occurred within 10-12 weeks of age, and those without the disease occurred within 12 weeks of age were excluded from the experimental group. In order to accelerate the early onset of the disease in the mouse model, all female NOD/LTJ mice without disease within 8 weeks of age were injected intraperitoneally with 150 mg/kg cyclophosphamide solution for injection (Jiangsu Hengrui Medicine Co., Ltd.), and injected again with the same dose of cyclophosphamide at the week 10 to accelerate the continuous and constant onset of diabetes mellitus, and the cyclophosphamide was prepared extemporaneously with normal saline (1 mg/ml normal saline) before the injection. After such treatment with cyclophosphamide, >70% of female NOD/LTJ mice typically developed overt diabetes within 11-12 weeks of age. Thus, the experimental needs of the DNA vaccine prevention and treatment were met. Abbott micro whole blood glucose tester was used to perform weekly micro whole blood glucose measurement on the NOD/LTJ mouse model of type 1 diabetes, type 1 diabetes was diagnosed if the blood glucose measurement is greater than or equal to 11.3-13.9 for twice.
[0055] Control group: 8-week-old female BALB/c normal mice (purchased from the Animal Center of the Military Medical Research Institute) were selected.
Example 2. Construction of Therapeutic DNA Vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 Eukaryotic Expression Vector
[0056] The upstream primer P1 containing a signal peptide and KpnI restriction site and the downstream primer P2 containing a XbaI restriction site were designed respectively. The primer sequences were as follows:
TABLE-US-00001 Upstream primer P1: (SEQ ID NO: 2) 5′-CGGGGTACCTGTTATGGATGGACTGAGTAACATTCTCTTTGTGATGGC CTTCCTGCTCTCTGGTGCTGCTCCTCTGAAGATTCAAG-3′
[0057] [wherein, the single-underlined part was the KpnI restriction site; the double-underlined part was the coding sequence of initiation codon and signal peptide]
TABLE-US-00002 Downstream primer P2: (SEQ ID NO: 3) 5′-GCTCTAGATTACTTCAGGTCCTCGCGCGGCGGTTTG-3′
[0058] [wherein, the single-underlined part was the XbaI restriction site]
[0059] Using the prokaryotic expression vector pRSETA-B7-2-PE40KDEL plasmid (constructed and preserved by our laboratory, see Chinese patent application 201010144610.0) as the template, high-fidelity Pfu DNA polymerase was used for PCR amplification. The amplification system was as follows:
TABLE-US-00003 2 × Pfu PCR Master Mix 10 μl P1 1 μl P2 1 μl pRSETA-B7-2-PE40KDEL(1:100) 2 μl ddH.sub.2O 6 μl Total volume 20 μl
[0060] The PCR reaction conditions were as follows: pre-denaturation at 96° C. for 5 min, denaturation at 96° C. for 1 min, annealing at 55° C. for 1 min, extension at 72° C. for 3 min, 30 cycles, and extension at 72° C. for 10 min. 1% agarose gel electrophoresis was used for identification.
[0061] The PCR amplification product was recovered, and the recovered product and the pcDNA3.1/Zeo(+) vector were double digested with KpnI+XbaI, and the digested products were recovered after electrophoresis. The enzyme digestion system was as follows:
TABLE-US-00004 10 × M buffer 2 μl Xba I 1 μl Kpn I 1 μl 0.1% BSA 2 μl Recovered PCR product or pcDNA3.1/Zeo(+) vector 10 μl ddH.sub.2O 4 μl Total volume 20 μl
[0062] After mixing, water bath at 37° C. for 3 h.
[0063] The digestion product was recovered by the steps as follows:
[0064] 1) The desired band was cut from the agarose gel with a clean blade and put into a 1.5 ml Ep tube.
[0065] 2) The gel was weighed, and a corresponding volume of sol solution was added in accordance with that for per 100 mg of gel, 300 μl of buffer QG was added.
[0066] 3) Incubation was carried out in a 50° C. water bath for 10 minutes until the gel was completely dissolved, during which turning upside down was carried out every 2-3 minutes to mix thoroughly. After the gel was completely dissolved, the color should change to yellow.
[0067] 4) 1 volume of isopropanol was added and mixed well.
[0068] 5) The sample was loaded to QIAquick column and centrifuged for 1 min. The waste solution was discarded, 0.5 ml of buffer QG was added, and centrifuged for 1 min.
[0069] 6) The waste solution was discarded, 0.75 ml of buffer PE was added, allowed to stand for 2 min to 5 min, and centrifuged for 1 min.
[0070] 7) The waste solution was discarded, and centrifugation was carried out for 1 min. The column was then placed in a clean 1.5 ml Ep tube.
[0071] 8) 30 μl of water for injection was added to the center of the column membrane, allowed to stand for 1 min, and then centrifuged for 1 min to collect the eluate.
[0072] The PCR product after double digestion was ligated to the pcDNA3.1/Zeo (+) vector, and the system was as follows:
TABLE-US-00005 10 × T4 Ligase buffer 2 μl T4 Ligase 2 μl Double digestion PCR product 10 μl Double digestion pcDNA3.1/Zeo(+) 3 μl ddH.sub.2O 3 μl Total volume 20 μl
[0073] The ligation tube was placed in an ice-water mixture to perform ligation at 4° C. overnight.
[0074] The ligation product was transformed into TOP10 competent bacteria according to the following method. The specific operations were as follows:
[0075] 1) 10 μl of ligation product and 20 μl of reagent A were diluted with sterile water to 100 μl, and kept on ice for later use.
[0076] 2) Transformation into TOP10 competent bacteria were performed on ice (5 min), and the above-mentioned diluted plasmid for later use was added.
[0077] 3) After staying on ice for 15 min, the TOP10 competent bacteria were allowed to stand at 37° C. for 1 min, coated on plate and incubated overnight at 37° C.
[0078] The positive colonies with Amp resistance were picked to extract plasmid, which was identified by double enzyme digestion (the conditions were the same as before), and the positive ones were sent to TaKaRa Company for sequencing confirmation.
[0079] The strains with the correct sequence identified were frozen and cultured in large quantities, and the PureYieild™ plasmid midi preparation kit was used to extract and purify the plasmid for DNA vaccine therapy on a large scale. The purified plasmid was dissolved in normal saline, 260/280>1.80, concentration>1.0 μg/μl.
[0080] In order to ensure the expression of B7-2-PE40 exotoxin fusion gene in eukaryotic cells, an eukaryotic expression vector pcDNA3.1/Zeo(+)-B7-2-PE40 containing B7-2-PE40 and Zeo resistance gene was constructed as shown in
Example 3. High-Level Expression of the Constructed Therapeutic DNA Vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 in Eukaryotic Cells
[0081] By using liposome-mediated method, 0.5×10.sup.5 to 2×10.sup.5 CHO-K1-RPE.40 cells were introduced into a 24-well plate (the medium was DMEM/F12, 7.5% FBS, 1× non-essential amino acids, and was added at 0.5 ml). 0.8 μg of the plasmid and 2.0 μl of Lipofectamine™2000 were diluted with 50 μl of OPTI-MEM I medium respectively, the two were mixed and incubated for 20 min, and then slowly added to the 24-well plate. After incubation with 5% CO.sub.2 at 37° C. for 6 h, the medium was replaced with complete DMEM medium. After 48 hours, the cultured cells and supernatant were collected and detected by RT-PCR and Western blotting.
[0082] RT-PCR Detection Steps:
[0083] 48 h after transfection, the grown CHO-K1-RPE.40 cells were washed twice with PBS, and the total cell RNA was extracted with Trizol kit according to the instructions, then reverse transcription synthesis of cDNA was performed, PCR amplification was carried out with the above primers P1 and P2 respectively, and β-actin was used as a reference control.
[0084] The reverse transcription system was as follows:
TABLE-US-00006 MgCl.sub.2 2 μl 10 × RNA PCR buffer 1 μl dNTP Mixture 1 μl RNase inhibitor 0.25 μl AMV 0.5 μl Oligo dT 0.5 μl RNA 4.75 μl Total volume 10 μl
[0085] The reaction conditions were: 42° C. for 30 min, 99° C. for 5 min, and 5° C. for 5 min.
[0086] The PCR reaction system was as follows:
TABLE-US-00007 MgCl.sub.2 3 μl 10 × LA buffer 4 μl ddH.sub.2O 31.75 μl LA Taq 0.25 μl P1 0.5 μl P2 0.5 μl RT reactant 10 μl Total volume 50 μl
[0087] The reaction conditions were as follows: pre-denaturation at 96° C. for 5 min, denaturation at 96° C. for 1 min, annealing at 55° C. for 1 min, extension at 72° C. for 3 min, 30 cycles, and extension at 72° C. for 10 min. 1% agarose gel electrophoresis was used for identification.
[0088] Human β-actin primer sequences were:
TABLE-US-00008 Upstream primer P5: (SEQ ID NO: 4) 5′ AGA AAA TCT GGC ACC ACA CC 3′ Downstream primer P6: (SEQ ID NO: 5) 5′ AGC ACT GTG TTG GCG TAC AG 3′
[0089] Western Blotting Detection Steps:
[0090] 1) Protein electrophoresis: 48 h after transfection of CHO-K1-RPE.40 cells, the culture supernatant was collected and concentrated using Amicon Ultra-4. 15 μl of the sample. The culture supernatant was taken and mixed evenly with 15 μl×SDS Loading buffer, boiled for 5 minutes, and subjected to SDS-PAGE protein electrophoresis, in which 80V electrophoresis was performed until the protein was electrophoresed out of the stacking gel, and then 160V electrophoresis was performed so that it reached the bottom of the separating gel, and the power supply was disconnected.
[0091] 2) Transfer membrane: Electrotransfer to PVDF membrane was performed. Immobilon-P was used as PVDF membrane, and immersed in methanol for 15 s, in water for 2 min, and in electrotransfer solution for 20 min; at the same time, filter paper and gel were immersed in electrotransfer solution for 15 min, according to +(white)/three-layer filter paper/membrane/gel/three-layer filter paper/black. The transfer membrane conditions were: 60 mA for 40 min.
[0092] 3) Blocking solution was used to block at room temperature for 2 h;
[0093] 4) Primary antibody diluted with blocking solution in an appropriate proportion, PEA polyclonal antibody or CD86 monoclonal antibody was added, and allowed to stand at 4° C. overnight;
[0094] 5) TBST was used to wash for 3 times, 5 min each time;
[0095] 6) HRP-anti-rabbit or mouse IgG secondary antibody diluted with blocking solution in appropriate proportion was added, and incubated at room temperature for 1 h;
[0096] 7) TBST was used to wash for 3 times, each 10 min;
[0097] 8) ECL method was used for exposure, development and fixation.
[0098] The protein electrophoresis formula of the above step 1) was as follows:
TABLE-US-00009 8% separating gel 5% stacking gel (5 ml) (2 ml) ddH.sub.2O 2.30 ml 1.40 ml 30% acrylamide 1.30 ml 0.33 ml 1.5M Tris-HCl(PH 8.8) 1.30 ml — 1.0M Tris-HCl(PH 6.8) — 0.25 ml 10% SDS 0.05 ml 0.02 ml 10% ammonium persulfate 0.05 ml 0.02 ml TEMED 0.003 ml 0.002 ml
[0099] The pcDNA3.1/Zeo(+)-B7-2-PE40 eukaryotic expression vector correctly identified by sequencing was transiently transfected into CHO-K1-RPE.40 cells, and the expression of B7-2-PE40 fusion protein was detected by the following methods: first, RT-PCR was used to detect the synthesis of B7-2-PE40 mRNA in the transfected cells, the results were shown in
TABLE-US-00010 TABLE 1 Detection of expression level of B7-2-PE40 exotoxin fusion protein Clone No. OD.sub.450 value Protein concentration (ug/L) Clone 12 0.624 0.21 Clone 13 0.615 0.20 Clone 14 0.767 0.27 Clone 15 0.709 0.25
Example 4. Therapeutic DNA Vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 Showing Sustained High Expression In Vivo and Highly-Efficient Targeted Killing Biological Effect
[0100] 75 to 150 μg of the purified therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was injected into the biceps femoris muscle of the right hind limb of normal Wistar rats. The mRNA of B7-2-PE40 could always be detected on day 2, 4, 7, 14, 21 and 28 after the injection, but could not be detected on day 56. The expression of B7-2-PE40 was highest on day 14 after the injection, and gradually decreased thereafter (
Example 5. Therapeutic DNA Vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 Showing Capacity of Effectively Reducing Blood Glucose Level in Subject with Type 1 Diabetes
[0101] Type 1 diabetes model NOD/LTJ mice were grouped for treatment and prevention (which was the same as in Example 5-8): they were divided into 4 groups in the experiment, respectively: (1) therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 treatment group; (2) therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 prevention group; (3) untreated control group; (4) methotrexate MTX (Jiangsu Hengrui Pharmaceutical Co., Ltd.) positive control group. Medication of the treatment group: From the eighth week when disease occurred in the type 1 diabetes model NOD/LTJ mice, the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was intramuscularly injected once every 2 weeks for a total of three treatments. Medication of the prevention group: Form the seventh week when no disease was found in the type 1 diabetes model NOD/LTJ mice, 100 ug of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was intramuscularly injected once every 2 weeks for a total of four treatments. The method of intramuscular injection combined with electrical pulse stimulation (200V/cm, 20 ms, 2 Hz, 8 pulses) was adopted. For the methotrexate MTX positive control group, started within 1 week after the new disease was diagnosed, intraperitoneal injection was carried out once a week, 1.5 mg˜3.0 mg/Kg for 10 consecutive weeks.
[0102] Treatment group: From the eighth week when disease was occurred in the type 1 diabetes model NOD/LTJ mice, 150 ug of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was intramuscularly injected once every 2 weeks for a total of three treatments. The results showed that the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 could not only effectively treat type 1 diabetes model NOD/LTJ mice, but also reduce blood glucose, and its effect was significantly better than that of methotrexate MTX positive control. Especially in the prevention group, from the seventh week when no disease was found in the type 1 diabetes model NOD/LTJ mice, 100 ug of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was intramuscularly injected once every 2 weeks for a total of four preventive medications. The therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 could very significantly prevent the occurrence and development of disease in the type 1 diabetes model NOD/LTJ mice, and maintain the blood glucose content at normal levels in the type 1 diabetes model NOD/LTJ mice (
[0103] Blood glucose monitoring step: Using an integrated micro whole blood glucose meter (purchased from Roche), weekly micro whole blood glucose measurement was performed on the above-mentioned type 1 diabetes model NOD/LTJ mice and balb/c normal mice. Diabetes was diagnosed when blood glucose was measured as >11.3-13.9 mmol/L for successive two times. The micro whole blood glucose measurement was performed weekly from week 8. Special attention was paid to the changes of blood glucose in the type 1 diabetes model NOD/LTJ mice after the treatment with the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40, in order to evaluate the therapeutic effect of the therapeutic DNA vaccine in the type 1 diabetes model NOD/LTJ mice.
[0104] The specific method of taking blood from tip of tail was as follows: this method of taking blood by cutting tail was adopted when the required blood volume was very small, in which the animal was fixed to expose rat tail, the tail was shaved to remove hair and then sterilized, and then immersed in warm water at about 45° C. for several minutes to fill the tail vessels; then the tail was wipe dried, and a sharp tool (knife or scissors) was used to cut off the tip of the tail, 0.3-0.5 cm, the integrated blood glucose meter was opened and a test strip was used to take blood spot, when the blood glucose meter emitted a signal sound, reading was carried out, the blood collection was over, and the wound was disinfected and pressed to stop bleeding.
Example 6. Therapeutic DNA Vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 Showing Capacity of Effectively Increasing Blood Insulin Level in Subject with Type 1 Diabetes
[0105] The serum insulin levels of the experimental group and the control group were detected by Elisa kit, and the detection was performed at different time periods, such as the 10.sup.th week in the early stage of the experiment, the 12.sup.th week in the early stage of the experiment, the 14.sup.th week in the middle stage of the experiment, and the 16.sup.th week in the late stage of the experiment. Special attention was paid to the changes of insulin level in the type 1 diabetes model NOD/LTJ mice after treatment with the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40, so as to evaluate the therapeutic effect of the therapeutic DNA vaccine in the type 1 diabetes model NOD/LTJ mice.
[0106] The method of taking blood from orbital venous plexus was as follows: after obtaining fresh blood, it was placed in an EDTA anticoagulant tube selected according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). The supernatant was collected carefully, and it should be centrifuged again if a precipitate formed during storage. The ELISA kit contained a 96-well plate that had been coated with antibodies, and the reaction results were determined by an enzyme-linked reaction apparatus. The concentrations of the standard substance were used as the abscissa and the OD values were used as the ordinate, thus the standard curve was drawn on graph paper, and the corresponding concentration was found out from the standard curve according to the OD value of the sample; it was then multiplied by its dilution factor; alternatively, the concentrations and OD values of the standard substance were used to calculate the linear regression equation of the standard curve, then the OD value of the sample was substituted into the equation to calculate the concentration of the sample, which was multiplied by its dilution factor to obtain the actual concentration of the sample.
[0107] Treatment group: From the eighth week when disease occurred in the type 1 diabetes model NOD/LTJ mice, 150 ug of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was intramuscularly injected once every 2 weeks for a total of three treatments. The results showed that the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 could effectively treat the NOD/LTJ mice, significantly increase the blood insulin content, and its effect was significantly better than that in the methotrexate MTX positive control (
Example 7. Therapeutic DNA Vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 Showing Capability of Effectively Reducing Concentration of Autoantibody ICA in Subject with Type 1 Diabetes
[0108] Treatment group: From the eighth week when disease occurred in the type 1 diabetes model NOD/LTJ mice, 150 ug of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was intramuscularly injected once every 2 weeks for a total of three treatments. Prevention group: From the seventh week when no disease was found in the type 1 diabetes model NOD/LTJ mice, 100 ug of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was intramuscularly injected once every 2 weeks for a total of four preventive medications. The change of serum ICA level in the type 1 diabetes model NOD/LTJ mice was detected by Elisa method. The results showed that the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 could not only effectively treat the occurrence and development of disease in the type 1 diabetes model NOD/LTJ mice, but also effectively reduce the blood concentration of ICA autoantibody in the type 1 diabetes model NOD/LTJ mice; both the treatment group and the prevention group showed the same effects, which were significantly better than that of the methotrexate MTX positive control (
Example 8. Therapeutic DNA Vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 Showing Capacity of Effectively Reducing Concentration of Autoantibody GAD in Subject with Type 1 Diabetes
[0109] Treatment group: From the eighth week when disease occurred in the type 1 diabetes model NOD/LTJ mice, 150 ug of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was intramuscularly injected once every 2 weeks for a total of three treatments. Prevention group: From the seventh week when no disease was found in the type 1 diabetes model NOD/LTJ mice, 100 ug of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was intramuscularly injected once every 2 weeks for a total of four preventive medications. The change of blood GAD level in the type 1 diabetes model NOD/LTJ mice was detected by Elisa method. The results showed that the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 could not only effectively treat the occurrence and development of disease in the type 1 diabetes model NOD/LTJ mice, but also significantly reduce the blood concentration of GAD autoantibody in the type 1 diabetes model NOD/LTJ mice, which were significantly better than that of the methotrexate MTX positive control, and especially remarkable for the prevention group (
[0110] The present invention has been described in detail above, and its purpose is to enable those skilled in the art to understand the content of the present invention and implement it, and not to limit the scope of protection of the present invention, all equivalent variations or modifications made according to the spirit of the present invention are intended to be included within the scope of the present invention.