WO2021068452A1 - 一种能有效治疗和/或预防1型糖尿病的dna疫苗及其用途 - Google Patents
一种能有效治疗和/或预防1型糖尿病的dna疫苗及其用途 Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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Definitions
- the invention relates to a DNA vaccine for the treatment of type 1 diabetes and its use, in particular to the use of a recombinant nucleic acid construct containing a B7-PE40 fusion gene for preparing a DNA vaccine for the treatment and/or prevention of type 1 diabetes.
- Type 1 diabetes is a refractory and heterogeneous metabolic disease and autoimmune disease that seriously endangers human health and life safety.
- the latest international big data research shows that since the beginning of the 21st century, the incidence of type 1 diabetes worldwide has continued to increase. Since 2010, Asia, Europe and North America have been growing at a rate of 4.0%, 3.2% and 5.3% respectively.
- the research on the epidemiology of type 1 diabetes in my country is not very late in nature.
- scientific research institutes also conducted general research and investigation on the epidemiology of type 1 diabetes in my country, but limited to the incomplete and imperfect research data at that time, the final statistical results and conclusions were only based on 22 Data reported by some hospitals in the city.
- the final research results show that there are at least 10 million patients with type 1 diabetes in my country, and the incidence of type 1 diabetes for all age groups is 1.01 per 100,000. Among them, the incidence rate for children aged 0-14 is 1.93 per 100,000, and those aged 30 and over The incidence rate is 0.69 per 100,000 people. In other words, there are about 13,000 new cases of type 1 diabetes in China every year, of which more than 9,000 cases are in people over 15 years old. Most new-onset type 1 diabetes is diagnosed in adulthood. The incidence of type 1 diabetes in children and adolescents aged 0-14 is significantly related to latitude. The incidence is higher in the north than in the south, but the incidence in people over 15 years of age has nothing to do with latitude. This may be related to genetic and environmental factors.
- NDDG National Diabetes Information Group
- ADA American Diabetes Association
- IDF-WPR International Diabetes Federation-Western Pacific Region
- WHO World Health Organization
- type 1 diabetes insulin-dependent diabetes
- 2 Type diabetes non-insulin dependent diabetes
- gestational diabetes other special types of diabetes.
- Type 1 diabetes was also called insulin-dependent diabetes. It is not only a refractory metabolic disease. From the perspective of its pathogenesis, it also belongs to the category of autoimmune diseases. Recently, the international Kirs also defines it as one of the important autoimmune diseases (IAIDs).
- IAIDs important autoimmune diseases
- the inventors discovered in the research that the recombinant nucleic acid construct pcDNA3.1/Zeo(+)-B7-2-PE40 transfected into eukaryotic cells is secreted into B7-2-PE40 after transcription, translation and post-translational modification. Extracellularly, pcDNA3.1/Zeo(+)-B7-2-PE40 can efficiently express B7-2-PE40 in eukaryotic cells and the expression product has good targeted immunosuppressive activity; the inventors also found that pcDNA3.
- the DNA vaccine of 1/Zeo(+)-B7-2-PE40 exotoxin fusion gene can effectively treat and/or prevent type 1 diabetes in rats. The present invention was completed based on these findings.
- One aspect of the present invention relates to the use of a recombinant nucleic acid construct containing a B7-2-PE40 exotoxin fusion gene in the preparation of a DNA vaccine for the treatment and/or prevention of type 1 diabetes.
- DNA vaccines can be used to treat and/or prevent mammalian subjects suffering from or at risk of developing type 1 diabetes.
- the sequence of the fusion gene is shown in SEQ ID NO:1.
- the recombinant nucleic acid construct contained in the DNA vaccine contains a B7-2-PE40 exotoxin fusion gene operatively linked to a recombinant expression vector, and the recombinant expression vector is selected from pcDNA3 .1/Zeo(+), pVAX1, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, pSVL and adenovirus vectors, preferably pcDNA3.1/Zeo(+).
- the sequence of the fusion gene is shown in SEQ ID NO:1.
- the DNA vaccine may also include a pharmaceutically acceptable immune adjuvant.
- the DNA vaccine is used for immunization by injection, mucosal, gene gun introduction, etc.; preferably, it is used for at least one selected from intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, etc. Immunization is carried out by injection, organ injection, thoracic injection and intraperitoneal injection.
- the DNA vaccine is an aqueous solution or a lyophilized powder injection for reconstitution that can be administered via injection or via mucosa.
- the present invention relates to the use of a recombinant nucleic acid construct containing the B7-2-PE40 exotoxin fusion gene in the preparation of a medicament for the treatment and/or prevention of type 1 diabetes.
- the sequence of the fusion gene is shown in SEQ ID NO:1.
- the present invention also relates to the use of a recombinant nucleic acid construct containing the B7-2-PE40 exotoxin fusion gene in the preparation of a kit or pharmaceutical composition, which can be used for treatment And/or prevent type 1 diabetes.
- a recombinant nucleic acid construct containing the B7-2-PE40 exotoxin fusion gene in the preparation of a kit or pharmaceutical composition, which can be used for treatment And/or prevent type 1 diabetes.
- the sequence of the fusion gene is shown in SEQ ID NO:1.
- the present invention relates to a method for the treatment and/or prevention of type 1 diabetes, comprising administering to a subject a therapeutically and/or preventively effective amount of a DNA vaccine containing a vector pcDNA3.1/Zeo (+)
- a DNA vaccine containing a vector pcDNA3.1/Zeo (+) The effectively linked B7-2-PE40 exotoxin fusion gene; preferably, the sequence of the B7-2-PE40 exotoxin fusion gene is shown in SEQ ID NO:1.
- the present invention provides a DNA vaccine for the treatment and/or prevention of type 1 diabetes, wherein the DNA vaccine contains B7-2-PE40 operably linked to the vector pcDNA3.1/Zeo(+) Exotoxin fusion gene; preferably, the sequence of the B7-2-PE40 exotoxin fusion gene is shown in SEQ ID NO:1.
- mammal refers to a subject who may suffer from, is about to suffer from, or has suffered from the autoimmune disease described in the present invention, including but not limited to domestic animals such as pigs, dogs, cats, cattle, sheep, etc. Humanity.
- the mammal is a human.
- prevention refers to the use of the DNA vaccine of the present invention to prevent the occurrence of the autoimmune disease when the mammal may or is about to occur the autoimmune disease.
- treatment refers to the use of the DNA vaccine of the present invention when the mammal has suffered or has developed the autoimmune disease to reduce, alleviate, delay, prevent, and eliminate the autoimmune disease. Or cure etc.
- PE40 exotoxin is an exotoxin commonly used in the art. It is produced by deleting the cell binding functional region Ia of Pseudomonas aeruginosa exotoxin A (PEA) with a molecular weight of 66kD and consisting of 613 amino acids. A protein with a molecular weight of 40kD.
- PEA Pseudomonas aeruginosa exotoxin A
- B7-2 used herein, also known as CD86, is a member of the immunoglobulin superfamily and is a natural ligand that expresses the two receptor molecules of the T cell surface costimulatory signal system CD28/CTLA4. B7-2 is constitutively expressed on the resting antigen-presenting cell APC, and once activated, it can be quickly expressed on B, T, monocyte-macrophages and dendritic cells. B7-2 is responsible for the initial stage of immune response.
- the costimulatory signal generated by the binding of B7-2 and its corresponding receptor not only plays an important role in regulating T cell activation and effector cytokine secretion, but also the B7-2/CD28 axis plays an important role in regulating T cell activation and effector cytokine secretion.
- the occurrence and development of autoimmune diseases such as type I diabetes and rheumatoid arthritis play a key role.
- B7-2-PE40 exotoxin fusion gene refers to the fusion gene of B7-2 and PE40 exotoxin.
- sequence of the fusion gene is shown in SEQ ID NO:1.
- recombinant nucleic acid construct contains the B7-2-PE40 exotoxin fusion gene and a recombinant expression vector operatively linked to it.
- the recombinant expression vector may be selected from pcDNA3.1/Zeo(+), pVAX1 , PWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, pSVL and adenovirus vector eukaryotic expression vector.
- the recombinant expression vector is pcDNA3.1/Zeo(+).
- GAD glutamate decarboxylase autoantibody
- glutamate decarboxylase autoantibody glutamate decarboxylase autoantibody
- Antibodies are autoantibodies produced by GAD-induced immune response and are an immunological indicator for the diagnosis of type 1 diabetes. The presence of GAD autoantibodies indicates a lack of endogenous insulin secretion.
- ICA islet cell autoantibody
- islet cell autoantibody is an important autoantibody closely related to the occurrence, development and transition of type 1 diabetes.
- Autoantibodies are organ-specific antibodies, and the antigen is islet cytoplasm.
- the components or microsomal components, mainly IgG, are a sign of pancreatic ⁇ -cell damage.
- the DNA vaccine of the present invention may be an aqueous solution or a lyophilized powder injection for reconstitution that can be administered via injection or via mucosa.
- the aqueous solution or lyophilized powder for reconstitution is prepared by aseptic operation techniques known in the art; and preferably, they are in a sterile state during storage, transportation, and use.
- the pharmaceutical composition can be any pharmaceutical form known in the art that can be used for administration, including pharmaceutical compositions, pharmaceutical preparations, kits and the like.
- the DNA vaccine involved in the use of the present invention can be administered by injection, mucosal and other routes, and these modes of administration are also part of the present invention.
- the most preferred route of administration suitable for the use of the present invention is parenteral route or administration via injection.
- the pharmaceutical composition is preferably a preparation for parenteral administration, including but not limited to a preparation for local injection and a preparation for systemic injection.
- the specific dosage form includes but not limited to a solution for injection. And injection powder.
- the medicament is a sterile aqueous solution for injection, or a sterile powder for injection that is reconstituted with water for injection before clinical use, especially a freeze-dried powder for injection.
- a freeze-dried powder injection it may also contain pharmaceutically acceptable excipients, such as mannitol.
- the DNA vaccine of the present invention can be introduced into a living body by a method known in the art.
- the introduction methods include, but are not limited to, intramuscular injection, gene gun introduction, mucosal immunization, intravenous injection, intraperitoneal injection, etc.
- introduction methods include, but are not limited to, intramuscular injection, gene gun introduction, mucosal immunization, intravenous injection, intraperitoneal injection, etc.
- Alpar HO et al. Expert Opin Drug Deliv, 2005, 2:829-842, which is incorporated herein by reference in its entirety.
- the daily dose may be administered to the subject all at once throughout the day, or the required dose may be divided into two, three, four or more small doses to be administered at appropriate intervals throughout the day.
- the small doses can be formulated into unit dosage forms, for example, each unit dosage form contains a corresponding amount of the total daily dose subdivided into appropriate times. Of course, it can also be administered in a certain period of time, such as once a day, once two days, once a week, once a month, once in February, once in March, once in June, once in a year, Apply once every two years and so on.
- DNA vaccines or pharmaceutical compositions of the present invention are used in specific clinical cases, their specific dosage may need to be changed correspondingly due to various factors, including but not limited to: Severity, age, sex, weight of the subject, route of administration, dosage form, etc.
- the present invention provides a new generation of vaccines or therapies that can specifically treat and/or prevent type 1 diabetes.
- the DNA vaccine can significantly reduce the blood sugar of patients with type 1 diabetes, effectively restore the patient’s own insulin secretion, and significantly reduce the pancreatic islet cells in the patient’s body.
- the content of autoantibodies (ICA) and glutamate decarboxylase autoantibodies (GAD) can effectively treat and/or prevent type 1 diabetes.
- the DNA vaccine can be highly expressed in the patient's body, and only subcutaneously or intramuscularly injected once, can maintain the curative effect for one month, greatly improve the compliance of the DNA vaccine medication, and effectively avoid the abuse of daily medication for type 1 diabetic patients.
- Figure 1 is a schematic diagram of constructing a therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 eukaryotic expression vector.
- Figure 2 shows the results of agarose electrophoresis analysis of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 amplified by PCR.
- lane 1 is the DNA marker
- lane 2 is the PCR amplification product of B7-2-PE40.
- Figure 3 shows the results of agarose electrophoresis analysis of recombinant plasmid pcDNA3.1/Zeo(+)-B7-2-PE40 by KpnI and XbaI double enzyme digestion.
- lane 1 is the DNA marker
- lane 2 is the recombinant plasmid digested with KpnI and XbaI.
- Figure 4 is a gene sequencing map of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 eukaryotic expression vector.
- Figure 5 shows the B7-2-PE40 determination sequence and open reading frame.
- Figure 6 is an agarose gel electrophoresis analysis diagram of the RT-PCR product after the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 eukaryotic expression vector is transfected into CHO-K1-RPE.40 cells.
- lane 1 is the DNA marker
- lane 2 is the amplification results of CHO-K1-RPE.40 cells B7-2-PE40 transfected with pcDNA3.1/B7-2-PE40
- lanes 3 and 4 are the results of transfection with pcDNA3.1 CHO-K1-RPE.40 cell expansion result with empty vector.
- Figure 7 shows the Western Blot detection of the target protein secreted by the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 in eukaryotic cells.
- lane 1 is transfection with pcDNA3.1/Zeo(+) empty vector
- lane 2 is transfection with pcDNA3.1/Zeo(+)-B7-2-PE40 vector.
- Fig. 8 shows that the eukaryotic cell expression product stably transfected with the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 has high-efficiency targeted killing biological activity on CD28 + Jurkat cells.
- Figure 9 shows the results of agarose electrophoresis analysis after intramuscular injection of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid.
- Figure 10 shows the clearance of CD28 + T cells in vivo after intramuscular injection of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid.
- Figure 11 shows the change rule of NOD/LTJ blood glucose in the treatment and prevention of type 1 diabetic mice with the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40.
- Figure 12 shows the change rule of NOD/LTJ blood insulin content in type 1 diabetic mice treated with therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40.
- Figure 13 shows the change rule of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 to prevent NOD/LTJ blood insulin levels in type 1 diabetic mice.
- Figure 14 shows the change rule of NOD/LTJ blood ICA autoantibodies in type 1 diabetic mice treated with therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40.
- Figure 15 shows the change rule of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 to prevent NOD/LTJ blood ICA autoantibodies in type 1 diabetic mice.
- Figure 16 shows the change rule of GAD autoantibodies in NOD/LTJ blood of type 1 diabetic mice treated by therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40.
- Figure 17 shows the change rule of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 to prevent NOD/LTJ blood GAD autoantibodies in type 1 diabetic mice.
- the expression vectors pcDNA3.1/Zeo(+), Zeocin TM , Trizol and Lipofectamine TM 2000 used in the examples were purchased from Invitrogen.
- Jurkat and Raji cell lines were purchased from ATCC (American Standard Biological Collection).
- CHO-K1-RPE.40 cell line Choinese hamster ovary cells resistant to Pseudomonas aeruginosa exotoxin cell line) was also purchased from ATCC in the United States; PEA polyclonal antibody, CD86 monoclonal antibody and TMB substrate color developing solution were purchased from Sigma.
- PVDF membrane and Amicon Ultra-4 were purchased from Millipore. ECL was purchased from Pierce Company.
- MTS CellTiter 96AQueous One Solution Cell Proliferation Assay
- PureYieild TM 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.
- the main instruments used in the examples are: 9700 PCR instrument (PerkinElmer), Ultraviolet detector (Beckman), MiniII protein electrophoresis instrument and protein semi-dry electroporation instrument (Bio-Rad), Gel-Pro3.1 gel imaging system (Media Cybemetic).
- Experimental group select the internationally recognized spontaneous type 1 diabetes mouse model NOD/LTJ (purchased from the Laboratory of Experimental Animals, Chinese Academy of Medical Sciences), and prepare with reference to conventional methods. Select 7-8 weeks old female NOD/LTJ mice, measure the tail tip micro blood every week from the 8th week of age, and observe the appearance of diabetic symptoms. Generally, the onset is 10-12 weeks old, and the experimental group is excluded when the onset does not occur in 12 weeks. In order to accelerate the early and massive onset of the mouse model as soon as possible, all female NOD/LTJ mice that were not onset at the age of 8 weeks were injected intraperitoneally with 150 mg/kg cyclophosphamide (Jiangsu Hengrui Pharmaceutical Co., Ltd.) injection.
- the same dose of cyclophosphamide was injected again to speed up the continuous and constant onset of diabetes.
- the cyclophosphamide was temporarily prepared with normal saline (1 mg/ml normal saline) before the injection. After such treatment with cyclophosphamide, generally >70% of female NOD/LTJ mice at 11-12 weeks of age will develop significant diabetes. So as to meet the experimental needs of DNA vaccine prevention and treatment.
- the Abbott Micro Whole Blood Glucose Meter was used to measure the micro whole blood glucose of type 1 diabetic mouse model NOD/LTJ every week. If the blood glucose was measured twice ⁇ 11.3-13.9 mmol/L, it was diagnosed as diabetes.
- Control group 8-week-old female BALB/c normal mice (purchased from the Animal Center of the Academy of Military Medicine) were selected.
- the primer sequence is as follows:
- Upstream primer P1 5'-CGG GGTACC TGTT ATGGATGGACTGAGTAACATTCTCTTTGT GATGGCCTTCCTGCTCTCTGGT GCTGCTCCTCTGAAGATTCAAG-3' (SEQ ID NO: 2)
- Downstream primer P2 5'-GC TCTAGA TTACTTCAGGTCCTCGCGCGGCGGTTTG-3' (SEQ ID NO: 3)
- 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 then extension at 72°C for 10 min. 1% agarose gel electrophoresis identification.
- the PCR amplified product was recovered, and the above recovered product and pcDNA3.1/Zeo(+) vector were digested with KpnI+XbaI, and the digested product was recovered after electrophoresis.
- the digestion system is as follows:
- the digested product is recovered according to the following steps:
- the ligation product was transformed into TOP10 competent bacteria according to the following method.
- the specific operations are as follows:
- the strains with the correct identified sequence are frozen and cultured in large quantities, and the PureYieild TM Plasmid Medium Extraction Kit is used to extract and purify the DNA vaccine therapeutic plasmid on a large scale.
- the purified plasmid was dissolved in physiological saline, 260/280 ⁇ 1.80, concentration>1.0 ⁇ g/ ⁇ l.
- a eukaryotic expression vector pcDNA3.1/Zeo(+)-B7-2-PE40 containing B7-2-PE40 and Zeo resistance gene Build as shown in Figure 1.
- the designed PCR primers were used to amplify human B7-2-PE40, and the amplified product was analyzed by agarose gel electrophoresis, and a specific band with an expected size of 1919 bp was seen (see Figure 2).
- the product recovered by double restriction digestion was cloned into the KpnI and XbaI restriction sites of the eukaryotic expression vector pcDNA3.1/Zeo(+) to construct a pcDNA3.1/Zeo(+)-B7-2-PE40 recombinant plasmid. Plasmid extraction, double enzyme digestion and electrophoresis (see Figure 3) and sequencing identification results ( Figure 4) yielded positive clones.
- Example 3 The constructed therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 is highly expressed in eukaryotic cells
- liposome-mediated method transfer 0.5 ⁇ 10 5 -2 ⁇ 10 5 CHO-K1-RPE.40 cells into a 24-well plate (medium is DMEM/F12, 7.5% FBS, 1 ⁇ non-essential amino acids), Medium 0.5ml. 0.8 ⁇ g plasmid and 2.0 ⁇ l Lipofectamine TM 2000 were respectively diluted with 50 ⁇ l OPTI-MEM I medium, the two were mixed and incubated for 20min, and then slowly added to the 24-well plate. After incubating at 37°C and 5% CO2 for 6 hours, it was replaced with complete DMEM medium. After 48h, the cultured cells and supernatant were collected and detected by RT-PCR and Western blotting.
- the reverse transcription system is as follows:
- the reaction conditions were 42°C for 30 minutes, 99°C for 5 minutes, and 5°C for 5 minutes.
- the PCR reaction system is as follows:
- reaction conditions are 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 identification.
- the sequence of human ⁇ -actin primer is:
- Upstream primer P5 5’ AGA AAA TCT GGC ACC ACA CC 3’ (SEQ ID NO: 4)
- Downstream primer P6 5’AGC ACT GTG TTG GCG TAC AG 3’ (SEQ ID NO: 5)
- Protein electrophoresis 48 hours after transfection of CHO-K1-RPE.40 cells, the culture supernatant was collected and concentrated using Amicon Ultra-4. Take 15 ⁇ l of the sample and mix it with 15 ⁇ l ⁇ SDS Loading buffer, boil for 5min, perform SDS-PAGE protein electrophoresis, 80V voltage electrophoresis until the protein swims out of the layered gel, then perform 160V electrophoresis to the bottom of the separation gel, disconnect the power supply.
- PVDF membrane Electric transfer to PVDF membrane.
- the PVDF membrane is Immobilon-P. Soak in methanol for 15 seconds, soak in water for 2 minutes, and soak in electrotransmission solution for 20 minutes. At the same time, add filter paper and gel to electrotransport solution for 15 minutes. Press + (white)/three-layer filter paper/membrane/glue/three-layer filter paper/black. Film transfer conditions: 60mA 40min.
- the protein electrophoresis formula of the above step 1) is as follows:
- the pcDNA3.1/Zeo(+)-B7-2-PE40 eukaryotic expression vector 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 method: Firstly, RT-PCR was used to detect the synthesis of B7-2-PE40mRNA in the transfected cells. The results are shown in Figure 6. CHO-K1-RPE.40 cells transfected with pcDNA3.1/Zeo(+)-B7-2-PE40 A specific band was seen at 1919bp, but this band did not appear in CHO-K1-RPE.40 cells transfected with an empty vector.
- the PCR amplification product of ⁇ -actin is an internal control.
- Western blotting was used to detect the antigenicity and secreted expression of the B7-2-PE40 fusion protein in the cell culture supernatant. The results show that no matter whether PEA polyclonal antibody or CD86 monoclonal antibody is used, there is a positive band at a relative molecular mass of about 90kDa, but the empty vector transfected cell supernatant does not show any bands.
- the results are shown in Figure 7.
- Among the stably transfected cells obtained by screening 1 ⁇ 10 6 stably transfected cells expressed approximately 0.23 ⁇ g/L of B7-2-PE40 exotoxin fusion protein in 24 hours ( Figure 8 and Table 1).
- Example 4 Therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 can be continuously expressed in vivo and has a highly effective targeted killing biological effect
- the purified 75-150 ⁇ g 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. After injection, 2d, 4d, 7d, 14d, The mRNA of B7-2-PE40 can be detected on both 21d and 28d, but it is almost undetectable at 56 days. The expression of B7-2-PE40 was highest at 14 days after injection, and then gradually decreased ( Figure 9). After intramuscular injection of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid, CD28 + T cells in the peripheral blood of rats were eliminated, as shown in Figure 10.
- Example 5 The therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 can effectively reduce the blood sugar content of type 1 diabetes
- Type 1 diabetes mouse model NOD/LTJ treatment and prevention groups (the same in Examples 5-8): The experiment is divided into 4 groups, namely: (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.
- Treatment group medication In the eighth week of onset of type 1 diabetes mouse model NOD/LTJ, the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was injected intramuscularly every 2 weeks for a total of three treatments.
- Medication of the prevention group In the seventh week of NOD/LTJ in the type 1 diabetic mouse model, 100ug of therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was injected intramuscularly every 2 weeks for a total of four treatments Times. Take the method of intramuscular injection combined with electrical pulse stimulation (200V/cm, 20ms, 2Hz, 8 pulses). The MTX positive control group of methotrexate was started within 1 week after the new disease was diagnosed, and was injected intraperitoneally once a week with 1.5mg ⁇ 3.0mg/Kg for continuous treatment for 10 weeks.
- Treatment group In the eighth week of onset of NOD/LTJ model in type 1 diabetic mice, 150ug of therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was injected intramuscularly every 2 weeks for a total of three treatments. The results show that the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 can not only effectively treat NOD/LTJ in type 1 diabetic mice, reduce blood sugar, and its effect is significantly better than the positive control of methotrexate MTX . Particularly prominent is the prevention group.
- Blood glucose monitoring steps using an integrated micro whole blood glucose meter (purchased from Roche), the above-mentioned type 1 diabetic mouse model NOD/LTJ and balb/c normal mice are measured weekly for micro whole blood glucose. Diabetes can be diagnosed when blood glucose is measured twice ⁇ 11.3-13.9mmol/L continuously. Micro whole blood glucose is measured once a week from the 8th week. Special attention is paid to the change of NOD/LTJ blood glucose in the type 1 diabetes mouse model after the treatment of the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 to evaluate the therapeutic DNA vaccine treatment of the type 1 diabetes mouse model NOD/ The efficacy of LTJ.
- the specific method of taking blood from the tip of the tail is as follows: cutting (trimming) the tail and collecting blood. Fix the animal and expose the tail of the rat. Cut off the tail hair and sterilize it, then immerse it in warm water at about 45°C for a few minutes to fill the tail blood vessels. Then dry the tail, use a sharp instrument (knife or scissors) to cut off the tip of the tail 0.3-0.5cm, open the integrated blood glucose meter with a test paper edge to stain the bleeding point, wait until the blood glucose meter emits a signal tone, read the blood, the blood sampling is over, the wound is disinfected and Compression to stop bleeding.
- Example 6 The therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 can effectively increase the blood insulin level of type 1 diabetes
- the serum insulin levels in the experimental group and the control group were detected by Elisa kit at different time periods such as the 10th week of the initial experiment, the 12th week of the early experiment, the 14th weekend of the middle experiment, and the 16th weekend of the late experiment. Pay special attention to the changes of NOD/LTJ insulin levels in type 1 diabetic mouse models after treatment with the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 to evaluate the therapeutic DNA vaccine treatment of type 1 diabetic mouse models NOD /LTJ's efficacy.
- the method of taking blood from the orbital venous plexus is as follows: After obtaining fresh blood, select an EDTA anticoagulant tube according to the requirements of the specimen, mix for 10-20 minutes, and centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
- the ELISA kit is a 96-well plate containing antibodies, and the reaction result is measured by an enzyme-linked reaction machine.
- Treatment group In the eighth week of onset of NOD/LTJ model in type 1 diabetic mice, 150ug of therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was injected intramuscularly every 2 weeks for a total of three treatments. The results show that the therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 can effectively treat the NOD/LTJ mouse model, increase the blood insulin content significantly, and its effect is significantly better than the positive control of methotrexate MTX ( Figure 12). Particularly prominent is the prevention group.
- Example 7 The therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 can effectively reduce the autoantibody ICA concentration of type 1 diabetes
- Treatment group Starting from the eighth week of onset of the NOD/LTJ model of type 1 diabetic mice, 150ug of therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was injected intramuscularly every 2 weeks for a total of three treatments.
- Prevention group In the seventh week of the NOD/LTJ type 1 diabetes mouse model without onset, 100ug of therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was injected intramuscularly every 2 weeks, and a total of four preventive medications Times. Elisa method was used to detect the changes of ICA level in NOD/LTJ serum of type 1 diabetes mouse model.
- Example 8 The therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 can effectively reduce the concentration of autoantibody GAD in type 1 diabetes
- Treatment group In the eighth week of the onset of NOD/LTJ in type 1 diabetic mice, 150ug of therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 plasmid was injected intramuscularly every 2 weeks for a total of three treatments.
- Prevention group In the seventh week of the NOD/LTJ type 1 diabetes mouse model without onset, 100ug of therapeutic DNA vaccine pcDNA3.1/Zeo(+)-B7-2-PE40 was injected intramuscularly every 2 weeks, and a total of four preventive medications Times. The level of GAD in NOD/LTJ serum of type 1 diabetic mouse model was detected by Elisa method.
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Abstract
提供了一种含有B7-2-PE40外毒素融合基因的重组核酸构建体在制备用于治疗和/或预防1型糖尿病的DNA疫苗或药物中的用途。该DNA疫苗可降低1型糖尿病患者的血糖,恢复患者自身胰岛素的分泌,减少患者体内胰岛细胞自身抗体(ICA)与谷氨酸脱羧酶自身抗体(GAD)的含量。
Description
本发明涉及一种治疗1型糖尿病的DNA疫苗及其用途,具体涉及含有B7-PE40融合基因的重组核酸构建体用于制备治疗和/或预防1型糖尿病的DNA疫苗的用途。
1型糖尿病是一种严重危害人类健康和生命安全的难治性、异质性的代谢性疾病和自身免疫性疾病。最新的国际大数据研究显示,进入21世纪以来,全世界1型糖尿病的发病率在持续不断的增多。自2010年开始,亚洲、欧洲和北美洲地区分别在以4.0%、3.2%和5.3%的速度增长。相比较而言,我国对1型糖尿病流行病学的研究起步实质上也并不很晚。早在20多年前即上世纪末科研单位也曾对我国1型糖尿病的流行病学进行过泛泛的研究调查,但限于当时研究数据的不全面不完善,最终统计结果和结论也仅仅是依据22个城市的部分医院自行上报的数据。为了确保我国1型糖尿病整体发病情况调查结果的准确性,由中华医学会糖尿病学分会前任主任委员翁建平教授领衔团队,联合我国13个地区的505家医院共同参与“2010年~2013年覆盖全年龄段的中国1型糖尿病研究”,历时3年的不懈努力,共获取1.33亿人的资料,调查范围约占中国总人口数的10%,包括6%的15岁以下儿童青少年。研究共收集到5018例新发1型糖尿病病例,其中65.3%的患者为20岁以上。最终研究结果显示,我国1型糖尿病患者至少近千万,全年龄段1型糖尿病发病率为1.01/10万人,其中0~14岁儿童发病率为1.93/10万人,30岁及以上人群发病率为0.69/10万人。也就是说,中国每年约有13000例新发1型糖尿病,其中超过9000例在15岁以上的人群中。大部分新发1型糖尿病则是在成年时才被诊断。0-14岁儿童青少年1型糖尿病发病率与纬度显著相关,北方比南方发病率高,但在15岁以上人群发病率与纬度变化无关。这可能与基因、环境因素有关。由此可见1型糖尿病在我国的发病率和发病总人数都远远高于既往的结论。该研究成果于2018年1月4日在线同期发表在国际著名权威医学期刊《英国医学杂志》和美国科学促进协会网站上。
随着对糖尿病病因和发病机制的不断研究和深入认识,使得人们对糖尿病的分型更加科学精准。在历经美国国家糖尿病资料组(NDDG)、美国糖尿病协会(ADA)、 国际糖尿病联盟-西太区(IDF-WPR)委员会及世界卫生组织(WHO)不断的修改完善,糖尿病的分型已从1936年单纯粗放的1型和2型分型法,最终演化成为世界各国所公认并正式采用的WHO糖尿病病因学分型法,即糖尿病可分为4型:1型糖尿病(胰岛素依赖型糖尿病)、2型糖尿病(非胰岛素依赖型糖尿病)、妊娠糖尿病及其它特殊类型糖尿病。依据国际上糖尿病最新病因分型原则,取消了“胰岛素依赖型糖尿病”和“非胰岛素依赖型糖尿病”原有分型名词,以避免混淆按治疗分型的概念。保留1型和2型分型名词,不再使用罗马数字I和Ⅱ。随着人类基因组学、分子生物学、人类遗传学、免疫病理学等学科的迅猛发展,特殊类型糖尿病又可分为8个亚类,亚类里还有更细的分支。迄今仅单基因糖尿病就发现了至少27种,而这些类型的糖尿病在以前都被误认为属于2型或1型。1型糖尿病曾又称之为胰岛素依赖型糖尿病,它不仅是一种难治性的代谢性疾病。从其发病机制的角度讲,它亦归属于自身免疫性疾病的范畴。新近国际学术界还将其定义为重要自身免疫性疾病(important autoimmune diseases,IAIDs)中的一类。
时至今日,国际上针对1型糖尿病的传统主流治疗策略始终是采用胰岛素或胰岛素类似物的长期补充疗法,这是确保1型糖尿病患者能有良好的生活质量和满意控制代谢水平的基础。然而,长期使用胰岛素常常会带来诸多的严重毒副作用,尤其是低血糖反应或低血糖昏迷以及胰岛素抗性等。近年来国际上有关1型糖尿病治疗新策略的研究和应用虽然有了长足的进展,诸如探索了全胰腺移植、胰岛细胞移植、成体干细胞或胚胎干细胞体外诱导分化胰岛β细胞的移植、基因治疗以及免疫抑制剂或免疫调节剂的应用等等,但都因为这样或那样种种条件的限制或技术上的不完善、不成熟,使得这些新疗法均未能在临床上形成主导性治疗方案或策略而被广泛应用。因此,如何能改善或克服上述药物或疗法所存在的弊端,抑或创造出新一代能特异的治疗1型糖尿病的药物或疗法已成为迫切需要解决的首要问题!近年来国际上有关1型糖尿病的治疗研究愈加倾向于针对1型糖尿病的发病机理,探索研制治疗性DNA疫苗的新策略则成为本领域关注的焦点与热点。2000、2008及2012年美国NIH先后三次都将有关重要自身免疫性疾病(包括1型糖尿病)的新型靶向性免疫抑制/调节剂尤其是治疗性DNA疫苗的研发列为重大课题中的重中之重进行优先资助,并向全美及欧共体国家公开招标。
发明内容
本发明人在研究中发现,转染入真核细胞的重组核酸构建体pcDNA3.1/Zeo(+)-B7-2-PE40经转录、翻译和翻译后修饰后B7-2-PE40被分泌到胞外,pcDNA3.1/Zeo(+)-B7-2-PE40可在真核细胞中高效表达B7-2-PE40并且表达产物具有很好的靶向免疫抑制活性;发明人还发现,pcDNA3.1/Zeo(+)-B7-2-PE40外毒素融合基因的DNA疫苗可以有效地治疗和/或预防大鼠1型糖尿病,本发明基于这些发现而得以完成。
因此,本发明提供的各个方面和特征概述如下:
本发明的一个方面,涉及一种含有B7-2-PE40外毒素融合基因的重组核酸构建体在制备用于治疗和/或预防1型糖尿病的DNA疫苗中的用途。此种DNA疫苗可以用于治疗和/或预防患有1型糖尿病或有罹患1型糖尿病风险的哺乳动物受试者。优选地,所述融合基因的序列如SEQ ID NO:1所示。
在本发明的一个具体实施方案中,所述DNA疫苗中所包含的重组核酸构建体,其含有与重组表达载体有效连接的B7-2-PE40外毒素融合基因,所述重组表达载体选自pcDNA3.1/Zeo(+)、pVAX1、pWLNEO、pSV2CAT、pOG44、pXT1、pSG、pSVK3、pBPV、pMSG、pSVL和腺病毒载体,优选为pcDNA3.1/Zeo(+)。优选地,所述融合基因的序列如SEQ ID NO:1所示。
在本发明中,所述的DNA疫苗还可以包含医药学上可接受的免疫佐剂。
在本发明的一个实施方案中,所述DNA疫苗用于以注射、粘膜、基因枪导入等方式实施免疫;优选的,其用于以至少一种选自静脉注射、动脉注射、肌肉注射、皮下注射、器官注射、胸腔注射和腹腔内注射的方式实施免疫。
在本发明的又一实施方案中,所述DNA疫苗为可经由注射或可经由粘膜施用的水溶液剂或复溶用冻干粉针剂。
在另一个方面,本发明涉及一种含有B7-2-PE40外毒素融合基因的重组核酸构建体在制备用于治疗和/或预防1型糖尿病的药物中的用途。优选地,所述融合基因的序列如SEQ ID NO:1所示。
在另一个方面,本发明还涉及一种含有B7-2-PE40外毒素融合基因的重组核酸构建体在制备试剂盒或药物组合物中的用途,所述试剂盒或药物组合物能够用于治疗和/或预防1型糖尿病。优选地,所述融合基因的序列如SEQ ID NO:1所示。
在又一个方面,本发明涉及一种治疗和/或预防1型糖尿病的方法,包括向受试者施用治疗和/或预防有效量的DNA疫苗,所述DNA疫苗含有与载体pcDNA3.1/Zeo(+) 有效连接的B7-2-PE40外毒素融合基因;优选地,其中所述B7-2-PE40外毒素融合基因的序列如SEQ ID NO:1所示。
在又一个方面,本发明提供了一种DNA疫苗,其用于治疗和/或预防1型糖尿病,其中所述DNA疫苗含有与载体pcDNA3.1/Zeo(+)有效连接的B7-2-PE40外毒素融合基因;优选地,其中所述B7-2-PE40外毒素融合基因的序列如SEQ ID NO:1所示。
本发明中相关术语的说明及解释
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。为了更好地理解本发明,下面提供相关术语的定义和解释。
用于本文的术语“哺乳动物”是指可能罹患、即将罹患、或已经罹患本发明所述自身免疫性疾病的受试者,其包括但不限于猪、狗、猫、牛、羊等家畜和人类。优选地,所述的哺乳动物是人类。
用于本文的术语“预防”是指所述哺乳动物处于可能发生、即将发生所述自身免疫性疾病的情况下使用本发明的DNA疫苗预防该自身免疫性疾病的发生。
用于本文的术语“治疗”是指所述哺乳动物已经罹患或已经发生所述自身免疫性疾病的情况下使用本发明的DNA疫苗,使该自身免疫性疾病减轻、缓解、延缓、阻止、消除或治愈等。
用于本文的术语“PE40外毒素”是本领域常用的外毒素,它是将分子量为66kD、由613个氨基酸组成的绿脓杆菌外毒素A(PEA)的细胞结合功能区Ia缺失后而产生一种分子量为40kD的蛋白质。
用于本文的术语“B7-2”又称CD86,属于免疫球蛋白超家族成员,是表达T细胞表面共刺激信号系统CD28/CTLA4两个受体分子的天然配体。B7-2构成性表达在静止的抗原提呈细胞APC上,一旦激活可快速表达在B、T、单核-巨噬细胞及树突状细胞上。B7-2主司免疫反应的初始期,B7-2与其相应受体结合所产生的共刺激信号不仅在调节T细胞活化及效应性细胞因子分泌中起重要作用,而且B7-2/CD28轴在自身免疫性疾病诸如I型糖尿病及类风湿关节炎的发生与发展发挥关键性作用。
用于本文的术语“B7-2-PE40外毒素融合基因”是指B7-2与PE40外毒素的融合基因。优选地,所述融合基因的序列如SEQ ID NO:1所示。
用于本文的术语“重组核酸构建体”,其含有B7-2-PE40外毒素融合基因和与其有效连接的重组表达载体,所述重组表达载体可选自pcDNA3.1/Zeo(+)、pVAX1、 pWLNEO、pSV2CAT、pOG44、pXT1、pSG、pSVK3、pBPV、pMSG、pSVL和腺病毒载体的真核表达载体。优选地,所述重组表达载体是pcDNA3.1/Zeo(+)。
用于本文的术语“GAD”为谷氨酸脱羧酶自身抗体(glutamate decarboxylase autoantibody),其是人体内抑制性神经递质γ-氨基丁酸的合成酶,主要存在于胰岛β细胞中,GAD自身抗体是GAD诱发免疫反应产生的自身抗体,是诊断1型糖尿病的免疫性指标,GAD自身抗体的存在,预示着内源性胰岛素分泌缺乏。
用于本文的术语“ICA”为胰岛细胞自身抗体(islet cell autoantibody),其是与1型糖尿病发生、发展及转轨紧密相关的重要自身抗体,自身抗体属器官特异性抗体,抗原为胰岛细胞浆成分或微粒体组分,主要为IgG类,是胰岛β细胞损伤的标志。
本发明的DNA疫苗可以是可经由注射或可经由粘膜施用的水溶液剂或复溶用冻干粉针剂。优选的,所述的水溶液剂或复溶用冻干粉针剂是通过本领域公知的无菌操作工艺制备的;并且还优选的,它们在贮藏、运输、使用过程中均呈无菌状态。
根据本发明,所述的药物组合物可以是本领域公知的任何可用于给药的药物形式,包括药物组合物、药物制剂、药盒等。虽然本发明所述用途中涉及的DNA疫苗可以经注射、粘膜等途径施用,并且这些施用方式也是本发明的一部分。但是,本领域技术人员清楚,适合于本发明所述用途的最优选施用途径是胃肠外途径或经注射方式施用。对于本发明的实施而言,所述的药物组合物优选是用于胃肠外给药的制剂,包括但不限于局部注射用制剂和全身注射用制剂,具体剂型包括但不限于注射用溶液剂和注射用粉针剂。更优选的,所述的药物是无菌的注射用含水溶液剂,或者是无菌的用于临床使用前用注射用水复溶配制的粉针剂,特别是冷冻干燥粉针剂。在制成冷冻干燥粉针剂时,其中还可以含有医药学上可接受的赋形剂,例如甘露醇。
本发明的DNA疫苗可以通过本领域公知的方法导入生物体内。所述的导入方法包括,但不限于,肌肉注射、基因枪导入法、③黏膜免疫、静脉注射法、腹腔注射法等,更详细的可以参考Alpar HO,et al.Expert Opin Drug Deliv,2005,2:829-842所公开的,该文献以其整体内容通过引用并入本文。
另外,根据下文中提供的研究结果,本领域技术人员可以容易地确定在其它哺乳动物中使用时有效剂量,特别是用于人时的有效剂量。可以将一天的剂量在一天中一次性全部给与受试者,也可以在一天内将需要的剂量分成两个、三个、四个或更多个小剂量以合适的间隔施用。所述小剂量可以配制成单元剂量形式,例如每个单元剂量形式含有日总剂量细分适宜次数的相应量。当然,也可以以一定的时间周期施用,例 如一天施用一次、两天施用一次、一周施用一次、一月施用一次、二月施用一次、三月施用一次、六月施用一次、一年施用一次、两年施用一次等。
本发明所述的DNA疫苗或药物组合物在用于具体的临床案例时,它们的具体使用量可因多种因素而可能需要作相应的变化,这些因素包括但不限于:受试者病况的严重程度,受试者的年龄、性别、体重,施用途径,药物剂型等等。
发明的有益效果
本发明提供了新一代能特异的治疗和/或预防1型糖尿病的疫苗或疗法,所述DNA疫苗可显著降低1型糖尿病患者的血糖,有效恢复患者自身胰岛素的分泌,明显减少患者体内胰岛细胞自身抗体(ICA)与谷氨酸脱羧酶自身抗体(GAD)的含量,从而达到有效的治疗和/或预防1型糖尿病。所述DNA疫苗可以在患者体内高效表达,仅皮下或肌肉注射一次,可维持疗效一个月,极大地提高了该DNA疫苗用药的依从性,有效地避免了1型糖尿病患者每天用药的弊端。
图1为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40真核表达载体构建示意图。
图2为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40经PCR扩增的琼脂糖电泳分析结果图。其中,泳道1为DNA marker;泳道2为B7-2-PE40的PCR扩增产物。
图3为重组质粒pcDNA3.1/Zeo(+)-B7-2-PE40经KpnⅠ和XbaⅠ双酶切的琼脂糖电泳分析结果图。其中,泳道1为DNA marker;泳道2为经KpnⅠ和XbaⅠ双酶切的重组质粒。
图4为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40真核表达载体基因测序图谱。
图5显示了B7-2-PE40测定序列及开放阅读框架。
图6为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40真核表达载体转染CHO-K1-RPE.40细胞后RT-PCR产物的琼脂糖凝胶电泳分析图。其中,泳道1为DNA marker;泳道2为转染pcDNA3.1/B7-2-PE40的CHO-K1-RPE.40细胞B7-2-PE40扩增结果;泳道3和4为转染pcDNA3.1空载体的CHO-K1-RPE.40细胞扩增结果。
图7为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40在真核细胞中分泌靶蛋白的Western Blot检测。其中,泳道1为转染pcDNA3.1/Zeo(+)空载体;泳道2为转染 pcDNA3.1/Zeo(+)-B7-2-PE40载体。
图8为稳定转染治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40的真核细胞表达产物对CD28
+Jurkat细胞具有高效靶向性杀伤生物活性。
图9为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40质粒肌肉注射后的琼脂糖电泳分析结果图。
图10为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40质粒肌肉注射后体内CD28
+T细胞的清除状况。
图11为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40治疗和预防1型糖尿病小鼠NOD/LTJ血糖的变化规律。
图12为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40治疗1型糖尿病小鼠NOD/LTJ血胰岛素含量的变化规律。
图13为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40预防1型糖尿病小鼠NOD/LTJ血胰岛素含量的变化规律。
图14为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40治疗1型糖尿病小鼠NOD/LTJ血ICA自身抗体的变化规律。
图15为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40预防1型糖尿病小鼠NOD/LTJ血ICA自身抗体的变化规律。
图16为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40治疗1型糖尿病小鼠NOD/LTJ血GAD自身抗体的变化规律。
图17为治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40预防1型糖尿病小鼠NOD/LTJ血GAD自身抗体的变化规律。
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
其中,实施例中所用到的表达载体pcDNA3.1/Zeo(+)、Zeocin
TM、Trizol及Lipofectamine
TM2000购自Invitrogen公司。Jurkat和Raji细胞系购自美国ATCC(美国标准生物品收藏中心)。CHO-K1-RPE.40细胞系(中国仓鼠卵巢细胞抗绿脓杆菌 外毒素细胞株)亦购自美国ATCC;PEA多抗、CD86单抗及TMB底物显色液购自Sigma公司。PVDF膜与Amicon Ultra-4购自Millipore公司。ECL购自Pierce公司。MTS(CellTiter 96AQueous One Solution Cell Proliferation Assay)及PureYieild
TM plasmid midiprep system购自Promega公司。KpnI酶、XbaI酶及T4连接酶购自TaKaRa公司。2×Pfu PCR MasterMix购自TIANGEN公司。QIAquick Gel Extraction Kit购自QIAGEN公司。
实施例中所用到的主要仪器有:9700 PCR仪(PerkinElmer),
紫外检测仪(Beckman),MiniII型蛋白电泳仪及蛋白半干电转仪(Bio-Rad),Gel-Pro3.1凝胶成像系统(Media Cybemetic)。
实施例1.1型糖尿病小鼠模型NOD/LTJ的建立
实验组:选用国际公认的自发性1型糖尿病小鼠模型NOD/LTJ(购自中国医学科学院实验动物所),参照常规方法进行制备。选用7-8周龄雌性NOD/LTJ小鼠,从第8周龄开始每周测定鼠尾尖微量血,并观察糖尿病症状出现。一般10-12周龄发病,当12周未发病就剔除出实验组。为了加速该鼠模型尽快在早期大量发病,取均为第8周龄未发病的雌性NOD/LTJ小鼠,用150mg/kg环磷酰胺(江苏恒瑞医药股份有限公司)注射液腹腔注射,在第10周时再进行一次同剂量环磷酰胺注射,以加快糖尿病的持续恒定发作,环磷酰胺在注射前用生理盐水临时配制(1mg/ml生理盐水)。采用环磷酰胺如此处理后,一般在第11-12周龄时>70%的雌性NOD/LTJ小鼠都会发生明显的糖尿病。从而能满足DNA疫苗防治的实验需求。采用雅培微量全血血糖测定仪,对1型糖尿病小鼠模型NOD/LTJ进行每周微量全血血糖测定,若血糖测定连续2次≧11.3-13.9mmol/L诊断为糖尿病。
对照组:选用8周龄雌性BALB/c正常小鼠(购自军事医学研究院动物中心)。
实施例2.治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40真核表达载体的构建
分别设计含有信号肽及KpnI酶切位点的上游引物P1,及含有XbaI酶切位点的下游引物P2。引物序列如下:
上游引物P1:5’-CGG
GGTACCTGTT
ATGGATGGACTGAGTAACATTCTCTTTGT
GATGGCCTTCCTGCTCTCTGGTGCTGCTCCTCTGAAGATTCAAG-3’(SEQ ID NO:2)
[其中,加单下划线的为Kpn I酶切位点;加双下划线的为起始密码子及信号肽编码序列] 下游引物P2:5’-GC
TCTAGATTACTTCAGGTCCTCGCGCGGCGGTTTG-3’(SEQ ID NO:3)
[其中,加单下划线的为Xba I酶切位点]
以原核表达载体pRSETA-B7-2-PE40KDEL质粒(由本实验室构建并保存,见中国专利申请201010144610.0)为模板,采用高保真的Pfu DNA聚合酶进行PCR扩增。扩增体系如下:
PCR反应条件如下:96℃预变性5min,96℃变性1min,55℃退火1min,72℃延伸3min,30个循环,后72℃延伸10min。1%琼脂糖凝胶电泳鉴定。
将PCR扩增产物回收,并用KpnI+XbaI双酶切上述回收产物及pcDNA3.1/Zeo(+)载体,电泳后回收酶切产物。酶切体系如下:
混匀后37℃水浴3h。
将酶切产物按如下步骤回收:
1)用干净刀片将目的条带从琼脂糖凝胶上切下放入1.5ml Ep管中。
2)称量凝胶的重量,按100mg的凝胶加300μl的缓冲剂QG加入相应体积的溶胶液。
3)50℃水浴孵育10min直至胶完全溶解,其间每2-3min上下颠倒以彻底混匀。胶完全溶解后,颜色应变为黄色。
4)加入1倍体积的异丙醇,充分混匀。
5)将样品加入QIAquick柱,离心1min。弃掉废液,加0.5ml缓冲剂QG,离心1min。
6)弃掉废液。加0.75ml的缓冲剂PE,放置2min-5min,离心1min。
7)弃掉废液,离心1min。后将柱放于1.5ml的干净Ep管中。
8)将30μl注射用水加入柱膜中央,放置1min后离心1min收集洗脱液。
将双酶切后的PCR产物与pcDNA3.1/Zeo(+)载体连接,体系如下:
将连接管至于4℃冰水混合物中连接过夜。
将连接产物按如下方法转化入TOP10感受态菌中。具体操作如下:
1)将连接产物10μl、试剂A 20μl用无菌水稀释至100μl,冰上备用。
2)冰上融化入TOP10感受态菌(5min),加入上述稀释备用质粒。
3)冰上放置15min,后37℃放置1min。铺板,37℃过夜。
挑取具有Amp抗性的阳性菌落,提取质粒,双酶切鉴定(条件同前),阳性者送TaKaRa公司测序确认。
将鉴定序列正确的菌株冻存并大量培养,采用PureYieild
TM质粒中提试剂盒大规模提取并纯化DNA疫苗治疗用质粒。提纯的质粒溶解于生理盐水中,260/280≥1.80,浓度>1.0μg/μl。
为使B7-2-PE40外毒素融合基因可在真核细胞中表达,一个含有B7-2-PE40以及Zeo抗性基因的真核表达载体pcDNA3.1/Zeo(+)-B7-2-PE40按图1所示构建。用所设计的PCR引物扩增人B7-2-PE40,扩增产物经琼脂糖凝胶电泳分析,可见预期大小为1919bp的特异性条带(见图2)。将双酶切回收后产物克隆入真核表达载体pcDNA3.1/Zeo(+)的KpnI和XbaI酶切位点,构建成pcDNA3.1/Zeo(+)-B7-2-PE40重组质粒,经质粒提取、双酶切后电泳(见图3)和测序鉴定结果(图4),得到阳性克隆。测序显示信号肽之后的碱基序列没有发生点突变和移码突变,与原核表达质粒pRSETA-B7-2-PE40KDEL中的B7-2-PE40基因序列完全一致(见图5),证实载体构建成功。
实施例3.构建的治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40在真核细胞中高效表达
采用脂质体介导方法,将0.5×10
5-2×10
5CHO-K1-RPE.40细胞传入24孔板(培养基为DMEM/F12,7.5%FBS,1×非必需氨基酸),培养基0.5ml。0.8μg质粒和2.0μl Lipofectamine
TM2000分别用50μl OPTI-MEM I培养基稀释,两者混合孵育20min,缓慢加入24孔板中。37℃、5%CO2孵育6h后,更换为完全DMEM培养基。48h后收集培养细胞及上清,采用RT-PCR及Western印迹进行检测。
RT-PCR检测步骤:
转染48h后,将生长的CHO-K1-RPE.40细胞用PBS清洗两次,参照说明书用Trizol试剂盒提取细胞总RNA,然后逆转录合成cDNA,分别用上述引物P1、P2进行PCR扩增,并采用β-actin作为参比对照。
反转录体系如下:
反应条件为42℃30min,99℃5min,5℃5min。
PCR反应体系如下:
反应条件如下:96℃预变性5min,96℃变性1min,55℃退火1min,72℃延伸3min,30个循环,后72℃延伸10min。1%琼脂糖凝胶电泳鉴定。
人β-actin引物序列为:
上游引物P5:5’AGA AAA TCT GGC ACC ACA CC 3’(SEQ ID NO:4)
下游引物P6:5’AGC ACT GTG TTG GCG TAC AG 3’(SEQ ID NO:5)
Western印迹检测步骤:
1)蛋白电泳:转染CHO-K1-RPE.40细胞48h后,收集培养上清并利用Amicon Ultra-4将其浓缩。取15μl样品与15μl×SDS Loading缓冲剂混匀,煮沸5min,行SDS-PAGE蛋白电泳,80V电压电泳至蛋白泳出积层胶,后行160V电泳至分离胶底部,断开电源。
2)转膜:电转至PVDF膜。PVDF膜选用Immobilon-P。在甲醇浸泡15s,水中泡2min,电转液中泡20min,同时将滤纸及胶泡电转液15min,按+(白色)/三层滤纸/膜/胶/三层滤纸/黑色。转膜条件:60mA 40min。
3)使用封闭液室温封闭2h;
4)加入以适当比例用封闭液稀释的一抗,PEA多抗或CD86单抗4℃过液;
5)TBST洗涤3次,每次5min;
6)加入以适当比例用封闭液稀释的HRP-抗兔或小鼠IgG二抗,室温孵育1h;
7)TBST洗涤3次,每次10min;
8)采用ECL方法曝光显影、定影。
以上步骤1)的蛋白电泳配方如下:
将测序鉴定正确的pcDNA3.1/Zeo(+)-B7-2-PE40真核表达载体瞬时转染CHO-K1-RPE.40细胞,采用以下方法检测了B7-2-PE40融合蛋白的表达:首先用RT-PCR检测转染细胞中B7-2-PE40mRNA的合成,结果如图6所示,转染 pcDNA3.1/Zeo(+)-B7-2-PE40的CHO-K1-RPE.40细胞在1919bp处可见特异性条带,而转染空载体的CHO-K1-RPE.40细胞未出现此条带。β-actin的PCR扩增产物为内参。采用Western印迹方法检测细胞培养上清B7-2-PE40融合蛋白的抗原性及分泌表达情况。结果表明无论采用PEA多克隆抗体还是采用CD86单克隆抗体,均在相对分子质量约90kDa处有阳性条带,而空载体转染细胞上清无任何条带显示,结果见图7。在所筛选获得的稳定转染细胞中,1×10
6个稳定转染细胞24h约表达0.23μg/L的B7-2-PE40外毒素融合蛋白(图8和表1)。
表1 B7-2-PE40外毒素融合蛋白表达水平检测
克隆号 | OD 450值 | 蛋白浓度(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 |
实施例4.治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40在体内可持续高表达并具有高效靶向性杀伤生物效应
将纯化的75-150μg治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40质粒注射入正常Wistar大鼠右后肢股二头肌内,在注射后2d、4d、7d、14d、21d及28d均可检测出B7-2-PE40的mRNA,56天时基本检测不到。B7-2-PE40的表达在注射后14天最高,此后逐渐减弱(图9)。肌肉注射治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40质粒后大鼠外周血中CD28
+T细胞被清除状况,详见图10。结果显示,T细胞清除状况与B7-2-PE40在体内的表达变化一致。在肌肉注射第2天,CD28
+T细胞已被明显杀伤,其比值低于正常及对照大鼠。治疗第4天时,CD28
+T细胞下降至最低,达34%。此后有短暂恢复。随着B7-2-PE40在14天表达高峰的到来,CD28
+T在21天时再一次下降至低谷。此后由于B7-2-PE40表达的降低,CD28
+T细胞所占比例逐渐增加,并于第56天时恢复至正常水平。
实施例5.治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40能有效降低1型糖尿病的血糖含量
1型糖尿病小鼠模型NOD/LTJ治疗及预防分组(实施例5-8均相同):实验分为4组,分别为:(1)治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40治疗组;(2)治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40预防组;(3)未治疗对照组;(4)甲氨蝶呤MTX(江苏恒瑞医药股份有限公司)阳性对照组。治疗组用药:在1型糖尿病小鼠模型NOD/LTJ发病第八周,每2周肌肉注射治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40一次,共治疗三次。预防组的用药:1型糖尿病小鼠模型NOD/LTJ未发病的第七周,每2周肌肉注射100ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40一次,共治疗四次。采取肌肉注射结合电脉冲刺激(200V/cm,20ms,2Hz,8脉冲)的方法。甲氨蝶呤MTX阳性对照组,在确诊新发病后1周内开始,每周腹腔内注射1次,1.5mg~3.0mg/Kg连续治疗10周。
治疗组:在1型糖尿病小鼠NOD/LTJ模型发病第八周,每2周肌肉注射150ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40质粒一次,共治疗三次。结果表明,治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40不仅能有效治疗1型糖尿病小鼠NOD/LTJ,使血糖降低,其效果明显优于甲氨蝶呤MTX阳性对照。尤为突出的是预防组,在1型糖尿病小鼠模型NOD/LTJ未发病的第七周,每2周肌肉注射100ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40一次,共预防用药四次。该治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40能非常显著地预防1型糖尿病小鼠NOD/LTJ的发生与发展,使1型糖尿病小鼠NOD/LTJ的血糖含量保持在正常水平(图11)。
血糖监测步骤:采用整合型微量全血血糖测定仪(购自罗氏),对上述1型糖尿病小鼠模型NOD/LTJ及balb/c正常小鼠进行每周微量全血血糖测定。当血糖测定连续2次≧11.3-13.9mmol/L时则可诊断为糖尿病。微量全血血糖测定从第8周开始每周一次。特别关注治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40治疗后1型糖尿病小鼠模型NOD/LTJ血糖的变化,以评判治疗性DNA疫苗治疗1型糖尿病小鼠模型NOD/LTJ的疗效。
通过尾尖取血的具体方法如下:割(剪)尾采血当所需血量很少时采用本法。固定动物并露出鼠尾。将尾部毛剪去后消毒,然后浸在45℃左右的温水中数分钟,使尾部血管充盈。再将尾擦干,用锐器(刀或剪刀)割去尾尖0.3-0.5cm,打开整合型血糖仪用试纸边沾出血点,等到血糖仪发出信号音后读数,采血结束,伤口消毒并压迫止血。
实施例6.治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40能有效增加1型糖尿病的血胰岛素水平
通过Elisa试剂盒检测实验组和对照组血清中胰岛素水平,分别在实验初期第10周、实验早期第12周、实验中期第14周末、实验晚期第16周末等不同时段进行。特别关注治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40治疗后1型糖尿病小鼠模型NOD/LTJ胰岛素水平的变化,以评判治疗性DNA疫苗治疗1型糖尿病小鼠模型NOD/LTJ的疗效。
采用眼眶静脉丛取血的方法具体如下:获取新鲜血液之后,根据标本的要求选择EDTA抗凝管,混合10-20分钟后,离心20分钟左右(2000-3000转/分)。仔细收集上清,保存过程中如有沉淀形成,应该再次离心。该ELISA试剂盒为已包抗体的96孔板,通过酶联反应仪测定反应结果。以标准物的浓度为横坐标,OD值为纵坐标,在坐标纸上绘出标准曲线,根据样品的OD值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。
治疗组:在1型糖尿病小鼠NOD/LTJ模型发病第八周,每2周肌肉注射150ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40质粒一次,共治疗三次。结果表明,治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40能有效治疗NOD/LTJ小鼠模型,使血胰岛素含量明显增加,其效果明显优于甲氨蝶呤MTX阳性对照(图12)。尤为突出的是预防组,在1型糖尿病小鼠模型NOD/LTJ未发病的第七周,每2周肌肉注射100ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40一次,共预防用药四次。该治疗性DNA疫苗能更加显著地使血胰岛素含量明显增加(图13)。
实施例7.治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40能有效降低1型糖尿病的自身抗体ICA浓度
治疗组:从1型糖尿病小鼠NOD/LTJ模型发病第八周开始,每2周肌肉注射150ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40质粒一次,共治疗三次。预防组:在1型糖尿病小鼠模型NOD/LTJ未发病的第七周,每2周肌肉注射100ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40一次,共预防用药四次。通过Elisa方法检测1型糖尿病小鼠模型NOD/LTJ血清中ICA的水平变化。结果表明,治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40不仅能有效治疗1型糖尿病小鼠NOD/LTJ的发生与发 展,而且还能有效地降低1型糖尿病小鼠NOD/LTJ血中ICA自身抗体浓度,治疗组与预防组的效果相同,均明显优于甲氨蝶呤MTX阳性对照(图14-15)。
实施例8.治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40能有效降低1型糖尿病的自身抗体GAD浓度
治疗组:在1型糖尿病小鼠NOD/LTJ发病第八周,每2周肌肉注射150ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40质粒一次,共治疗三次。预防组:在1型糖尿病小鼠模型NOD/LTJ未发病的第七周,每2周肌肉注射100ug治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40一次,共预防用药四次。通过Elisa方法检测1型糖尿病小鼠模型NOD/LTJ血清中GAD的水平变化。结果表明,治疗性DNA疫苗pcDNA3.1/Zeo(+)-B7-2-PE40不仅能有效治疗1型糖尿病小鼠NOD/LTJ的发生与发展,而且还能显著地降低1型糖尿病小鼠NOD/LTJ血中GAD自身抗体浓度,明显优于甲氨蝶呤MTX阳性对照,尤其是在预防组效果十分突出(图16-17)。
以上对本发明做了详尽的描述,其目的在于让本领域技术人员能够了解本发明的内容并加以实施,并不能以此限制本发明的保护范围,凡根据本发明的精神实质所做的等效变化或修饰都应涵盖在本发明的范围内。
Claims (10)
- 包含B7-2-PE40外毒素融合基因的重组核酸构建体在制备用于治疗和/或预防1型糖尿病的DNA疫苗中的用途。
- 根据权利要求1所述的用途,其中所述B7-2-PE40外毒素融合基因与重组表达载体有效连接,所述重组表达载体选自pcDNA3.1/Zeo(+)、pVAX1、pWLNEO、pSV2CAT、pOG44、pXT1、pSG、pSVK3、pBPV、pMSG、pSVL和腺病毒载体,优选地,所述重组表达载体是pcDNA3.1/Zeo(+)。
- 根据权利要求1或2所述的用途,其中所述B7-2-PE40外毒素融合基因的序列如SEQ ID NO:1所示。
- 根据权利要求1-3任一项所述的用途,其中所述DNA疫苗还包含医药学上可接受的免疫佐剂。
- 根据权利要求1-4任一项所述的用途,其中所述DNA疫苗用于以注射、粘膜、基因枪导入等方式实施免疫;优选的,其用于以至少一种选自静脉注射、动脉注射、肌肉注射、皮下注射、器官注射、胸腔注射和腹腔内注射的方式实施免疫。
- 根据权利要求1-4任一项的用途,其中所述DNA疫苗为经由注射或经由粘膜施用的水溶液剂或复溶用冻干粉针剂。
- 含有B7-2-PE40外毒素融合基因的重组核酸构建体在制备用于治疗和/或预防1型糖尿病的药物组合物中的用途。
- 根据权利要求7所述的用途,其中所述B7-2-PE40外毒素融合基因与重组表达载体有效连接,所述重组表达载体选自pcDNA3.1/Zeo(+)、pVAX1、pWLNEO、pSV2CAT、pOG44、pXT1、pSG、pSVK3、pBPV、pMSG、pSVL和腺病毒载体,优选地,所述重组表达载体是pcDNA3.1/Zeo(+)。
- 根据权利要求7或8所述的用途,其中所述B7-2-PE40外毒素融合基因的序列如SEQ ID NO:1所示。
- 根据权利要求7-9任一项所述的用途,其中所述药物组合物还包含医药学上可接受的载体。
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