WO2020037809A1 - 蓖麻毒素b的制备及其应用 - Google Patents

蓖麻毒素b的制备及其应用 Download PDF

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WO2020037809A1
WO2020037809A1 PCT/CN2018/111237 CN2018111237W WO2020037809A1 WO 2020037809 A1 WO2020037809 A1 WO 2020037809A1 CN 2018111237 W CN2018111237 W CN 2018111237W WO 2020037809 A1 WO2020037809 A1 WO 2020037809A1
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protein
diabetes
type
rats
protein according
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侯峰
刘文森
许娜
曹利利
宫鹏涛
李思明
陈星远
丁鹤
王典
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侯峰
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

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  • the invention relates to the technical field of anti-diabetic protein drugs, in particular to a recombinant ricin B chain protein mutant for type 2 diabetes.
  • T2DM type 2 diabetes mellitus
  • the drugs used to treat T2DM at this stage are mainly traditional anti-diabetic drugs, including: sulfonylureas, glenalides, biguanides, thiazolidinediones, ⁇ -glucosidase inhibitors, and insulin, but these drugs are different. Degree of adverse reactions, such as hypoglycemia, gastrointestinal discomfort, obesity and so on. Therefore, it is particularly necessary to develop new and better drugs for the treatment of diabetes. With the continuous research on the basic theory of diabetes, the development of new anti-diabetic drugs has become a hot topic at home and abroad. Many pharmaceutical companies are developing new anti-diabetic drugs to meet current diabetes treatment needs.
  • Ricin is a heterodimeric glycoprotein that is linked by two chains, the A chain (RTA) and the B chain (RTB), through a disulfide bond.
  • RTA is a ribosome inactivating protein that can inhibit protein synthesis in mammalian cells.
  • RTB is a two-leaf-shaped molecule composed of two identically folded topological globular domains, each of which includes three subregions ( ⁇ , ⁇ , ⁇ ), and only 1 ⁇ and 2 ⁇ have significant galactose binding. Active, non-toxic, with lectin activity, can assist the A chain to enter the cell to play biological functions.
  • RTB can combine different sugar structures to regulate various biological processes, including cell-host interactions, pathogens, and innate immune responses.
  • the present invention provides a recombinant ricin B chain protein mutant and its expression method, and verifies its hypoglycemic effect on rats with type 2 diabetes .
  • mutants including insertion, deletion, replacement of a protein comprising the SEQ ID: 1 sequence in the preparation of a medicine for treating type 2 diabetes.
  • the coding nucleotide of the SEQ ID: 1 protein sequence is SEQ ID: 2.
  • the protein of SEQ ID: 1 can reduce fasting blood glucose levels in rats with type 2 diabetes.
  • the protein of SEQ ID: 1 can reduce the level of glycated hemoglobin in rats with type 2 diabetes.
  • the protein of SEQ ID: 1 can reduce drinking water in rats with type 2 diabetes.
  • the protein of SEQ ID: 1 can increase body weight and improve weight loss in rats with type 2 diabetes.
  • the protein of SEQ ID: 1 can improve triglyceride (TG) and total cholesterol (TCH) levels in rats with type 2 diabetes.
  • the protein of SEQ ID: 1 can improve glucose tolerance in rats with type 2 diabetes.
  • the method for preparing a protein of SEQ ID: 1 is prepared by an E. coli expression system.
  • the vector used in the E. coli expression system is a PET series vector, and the expression strain used is BL21.
  • the invention provides a method for expressing a recombinant ricin B chain protein mutant, and expressing the recombinant ricin B chain protein mutant through an E. coli expression system.
  • the invention also provides the application of the recombinant ricin B chain protein mutant against type 2 diabetic rats.
  • the beneficial effect of the present invention is that the recombinant ricin toxin B chain protein mutant of the present invention can improve the weight and water consumption of type 2 diabetic rats, and also has the effect of significantly lowering blood sugar and blood lipids.
  • the glucose tolerance test proves that the protein It has therapeutic effect on type 2 diabetic rats.
  • the invention provides the application of the recombinant ricin B chain protein mutant against type 2 diabetes, and the experiment of the recombinant ricin B chain protein mutant on type 2 diabetic rats proves that the recombinant ricin B chain protein of the invention Mutants can improve type 2 diabetes.
  • rRTB ricin B chain
  • tRTB is used as a template.
  • PCR amplification was performed with tRTB anti-sense primer, and the obtained PCR product was ligated to pMD19-T simple cloning vector (Bao Biological Engineering (Dalian) Co., Ltd.) to obtain pMD19T-tRTB. It was linked with the PET-28a expression vector digested with the same NdeI and HindIII restriction enzymes. The resulting linked product was transferred into E. coli competent cells BL21 (DE3).
  • the positive bacteria were screened to obtain positive plasmids.
  • the plasmids were extracted from the positive bacteria and sequenced.
  • the plasmid was a plasmid obtained by inserting Sequence 1 in the sequence listing between the NdeI and HindIII restriction sites of the PET28a expression vector. This plasmid was named PET28a-tRTB, and the corresponding positive bacteria was named BL21 (DE3) / PET28a-tRTB.
  • the positive bacteria BL21 (DE3) / PET28a-tRTB obtained above were inoculated at a ratio of 1: 100v / v into 5 mL of Kan + (concentration: 50 ⁇ g / mL) TB medium, and maintained at a constant temperature of 180 r / min at 37 ° C.
  • IPTG was added to the induction group to a final concentration of 1 mmol / L, the same as the above culture conditions, that is, induction at 37 ° C and 180r / min constant temperature shaking for 10h; after induction for 10h, at 4 ° C, 8000r Centrifuge / min to obtain bacterial cell pellet.
  • the obtained bacterial cell pellet was resuspended by sonication, and the inclusion body pellet was obtained by centrifugation. After 12% SDS-PAGE electrophoresis analysis, the recombinant ricin B chain protein was specifically expressed in the inclusion body, and its molecular weight was It is 38Kda, which is consistent with the expected protein size.
  • the inclusion body lysate obtained above was subjected to affinity chromatography. When the imidazole concentration was 300 mmol / L, the elution peak was collected to be the purified recombinant ricin B chain protein. Purified recombinant ricin B chain protein was renatured by dialysis gradient. After the renaturation was completed, the protein was concentrated with PEG20000, and the protein concentration was measured by BCA method. The protein was sterilized by filtration through a 0.22 ⁇ m filter and stored at -80 ° C.
  • mice Ten male wistar rats were randomly selected as the normal control group. After the other rats were fed with high-fat diet for 4W, the remaining single small dose (30mg / kg) was intraperitoneally injected with STZ to induce type 2 diabetes model (STZ dissolved in In the citric acid-sodium citrate buffer solution, the injection was completed within half an hour). After 1W, fasting but not water for 12h, take blood from the tail vein, and use a blood glucose meter to measure fasting blood glucose (FBG), where FBG ⁇ 7.8mmol / One person succeeded in a type 2 diabetes model. The test animals were fed in an animal center in a standardized feeding room with alternating light and dark for 12 hours. The room temperature was 20-25 ° C, the relative humidity in the room was 45% -60%, and the feeding room was well ventilated. During the test, normal pellets and high fat were given. Sugar feed, experimental animals drink freely.
  • STZ type 2 diabetes model
  • Fifty successful rats were selected and randomly divided into 5 groups: type 2 diabetes model group, positive drug control group, high protein concentration group, medium protein concentration group and low protein concentration group.
  • the drug in the positive control group was metformin hydrochloride, with a concentration of 200 mg / kg, and the protein administration group was given 400 ug / kg, 300 ug / kg, and 200 ug / kg of protein by intraperitoneal injection.
  • Normal control group and model group were injected with equal volume of purified water intraperitoneally, protein was administered once a week, metformin was administered once a day, and 6W was continuously administered.
  • standard pellets of normal feed, high-fat and high-sugar feed were given, and the test animals were free to drink water.
  • fasting blood glucose value was measured by blood glucose meter.
  • fasting blood glucose was measured.
  • Glucose was measured by intraperitoneal injection of glucose at 0, 30, 60, 90, and 120 minutes. Rats were fasted for 8 hours and killed after 2 days. Blood was taken from the abdominal aorta at 1.5 ml, 3000 r / min x 5 min x 2, and serum and sinking red blood cells were taken after centrifugation. The serum taken by rats is mainly used for the detection of biochemical indicators.
  • the results are shown in Table 1.
  • the drinking water and weight of type 2 diabetic rats were significantly improved compared with the model group after the RTB mutant intervention.
  • FBG fasting blood glucose
  • GHb glycated hemoglobin
  • Table 3 RTB protein has a better effect on improving triglycerides (TG) and total cholesterol (TCH); as shown in Table 4, glucose tolerance experiments have shown that RTB has a higher improvement Effect of glucose tolerance levels in type 2 diabetes.

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Abstract

涉及抗糖尿病药物的技术领域，具体为一种用于治疗2型糖尿病的重组蛋白药物，其能降低2型糖尿病大鼠的血糖和血脂。

Description

蓖麻毒素B的制备及其应用 技术领域
本发明涉及抗糖尿病蛋白药物技术领域，具体为一种用于对2型糖尿病的重组蓖麻毒素B链蛋白突变体。
背景技术
糖尿病是一种因胰岛素分泌不足和或胰岛素抵抗而引起的糖、脂肪和蛋白质代谢紊乱的免疫系统疾病，涉及全身各个系统，甚至可诱发许多致命性并发症，严重危害人类健康，糖尿病已经成为当前危害人类健康的第三大疾病。90％以上的糖尿病患者为2型糖尿病(type 2diabetes mellitus，T2DM)。目前，全世界约有2.46亿人患有T2DM，预计在20年内T2DM患者数目将增至3.8亿。中国是全球糖尿病患者人数最多的国家，但中国T2DM患者的血糖达标率严重不足。现阶段治疗T2DM的药物主要为传统抗糖尿病药物，包括：磺酰脲类、格列奈类、双胍类、噻唑烷二酮、α-葡萄糖苷酶抑制剂及胰岛素等，但这些药物均存在不同程度的不良反应，如引发低血糖、胃肠道不适、肥胖等。因而开发更优良的糖尿病治疗新药显得尤为必要，随着对糖尿病基础理论研究的不断深入，抗糖尿病新药的开发成为国内外研究的热点。许多制药公司正在开发新的抗糖尿病药物，以满足目前的糖尿病治疗需求。
由于糖尿病复杂的发病机制，使得医学工作者在药物研发的过程中，逐渐发现了许多潜在的抗糖尿病疗法的新型目标。与此同时，据统计，2013至今进入Ⅲ期临床试验或递交NDA/BLA或获得批准用于治疗糖尿病的药 物有65种，分为非胰岛素类抗糖尿病药(41种，包括用于治疗糖尿病相关疾病的药物10种，占63.1％)、胰岛素类药(8种，12.3％)和复方制剂(16种，24.6％)。这些数据表明，国际制药公司将开发糖尿病新药的重点集中在非胰岛素类抗糖尿病药物上，本发明重组蓖麻毒素B链蛋白在一定程度上契合国际药物开发的需求，应用前景广阔。
蓖麻毒素(Ricin)是一种由A链(RTA)和B链(RTB)两条链通过二硫键连接的异源二聚体糖蛋白。其中，RTA是一种在哺乳动物细胞内可抑制蛋白质合成的核糖体灭活蛋白。RTB是一个两叶形结构的分子，由两个相同折叠的拓扑学球状结构域构成，每一个区域又包括三个亚区(ɑ，β，γ)，只有1ɑ和2β有明显的半乳糖结合活性，无毒，具有凝集素活性，可协助A链进入细胞内发挥生物学功能。RTB可以结合不同的糖结构，调节各种生物过程，包括细胞与宿主、病原体相互作用和先天免疫反应。
目前，通过国内外公开出版物的检索并未发现重组蓖麻毒素B链蛋白突变体治疗2型糖尿病以及并发症的相关报道。
发明内容
为了解决目前治疗2型糖尿病药物效果不理想，副作用较多的问题，本发明提供一种重组蓖麻毒素B链蛋白突变体及其表达方法，并验证其对2型糖尿病大鼠的降糖效果。
本发明的技术方案为：
包含SEQ ID：1序列的蛋白在制备治疗2型糖尿病药物中的应用。
包含SEQ ID：1序列的蛋白的插入、删除、替换等突变体在制备治疗2 型糖尿病药物中的应用。
所述SEQ ID：1蛋白序列的编码核苷酸为SEQ ID：2。
SEQ ID：1序列的蛋白可降低2型糖尿病模型的大鼠空腹血糖水平。
SEQ ID：1序列的蛋白可降低2型糖尿病模型的大鼠糖化血红蛋白水平。
SEQ ID：1序列的蛋白可降低2型糖尿病模型的大鼠的饮水量。
SEQ ID：1序列的蛋白可增加2型糖尿病模型的大鼠的体重，改善消瘦症状。
SEQ ID：1序列的蛋白可改善2型糖尿病模型的大鼠的甘油三酯(TG)和总胆固醇(TCH)水平。
SEQ ID：1序列的蛋白可改善2型糖尿病模型的大鼠的耐糖能力。
SEQ ID：1序列的蛋白的制备方法，通过大肠杆菌表达系统制备。
所述大肠杆菌表达系统所用载体为PET系列载体，所用表达菌种为BL21。
本发明提供了一种重组蓖麻毒素B链蛋白突变体的表达方法，通过大肠杆菌表达系统表达重组蓖麻毒素B链蛋白突变体。
本发明还提供了重组蓖麻毒素B链蛋白突变体抗2型糖尿病大鼠的应用。
本发明的有益效果是：本发明的重组蓖麻毒素B链蛋白突变体具有改善2型糖尿病大鼠的体重和饮水量，同时还具有显著的降低血糖和血脂的效果，糖耐量实验证明该蛋白对2型糖尿病大鼠有治疗作用。
具体实施方式
本发明提供了重组蓖麻毒素B链蛋白突变体抗2型糖尿病的应用，通过 重组蓖麻毒素B链蛋白突变体对2型糖尿病大鼠的实验证明：本发明的重组蓖麻毒素B链蛋白突变体可以改善2型糖尿病。
实施例1重组蓖麻毒素B链蛋白突变体制备
1大肠杆菌表达工程菌BL21(DE3)/PET28a-tRTB的获得
在氨基酸序列不变的条件下，根据已有的蓖麻毒素B链(rRTB)突变体进行密码子优化，根据上述的蓖麻毒素B链密码子优化后的全序列作为模板，用tRTB sense primer和tRTB anti-sense primer进行PCR扩增，获得的PCR产物连接至pMD19-T simple克隆载体(宝生物工程(大连)有限公司)，获得pMD19T-tRTB。与经过同样NdeI与HindIII限制性酶切的PET-28a表达载体链接，得到的链接产物转入大肠杆菌感受态细胞BL21(DE3)，筛选获得阳性菌，对阳性菌提取质粒后进行测序，结果该质粒为序列表中序列1插入PET28a表达载体的NdeI与HindIII的酶切位点间得到的质粒，将该质粒命名为PET28a-tRTB，其对应的阳性菌命名为BL21(DE3)/PET28a-tRTB。
2重组蓖麻毒素B链蛋白突变体的诱导表达与纯化
将上述获得的阳性菌BL21(DE3)/PET28a-tRTB按1：100v/v的比例接种于5mL含Kan +(浓度为50μg/mL)的TB培养基中，在37℃下，180r/min恒温振荡培养至OD 600＝0.6时，诱导组添加IPTG至终浓度为1mmol/L，同上述培养条件即在37℃下，180r/min恒温振荡诱导10h；经诱导10h后，在4℃下，8000r/min离心获得菌体沉淀。
获得的菌体沉淀重悬用超声破碎方法将其裂解，离心获得包涵体沉淀；经12％SDS-PAGE电泳分析，重组蓖麻毒素B链蛋白在包涵体有特异性的表达，且其蛋白分子量为38Kda，与预期的蛋白大小相符合。将上述获得的 包涵体溶解液进行亲和层析，当咪唑浓度为300mmol/L时，收集洗脱峰即为纯化后的重组蓖麻毒素B链蛋白。纯化后的重组蓖麻毒素B链蛋白进行透析梯度复性。复性结束后用PEG20000浓缩蛋白，BCA法测定蛋白浓度，用0.22μm滤器过滤除菌后保存于-80℃。
实例2重组蓖麻毒素B链蛋白突变体抗2型糖尿病大鼠效果实验
1实验动物和分组
1.1模型的制作
试验用的雄性wistar大鼠，随机留取10只作为正常对照组，其他大鼠高脂饲料喂养4W后，其余单次小剂量(30mg/kg)腹腔注射STZ诱导2型糖尿病模型(STZ溶解在柠檬酸-柠檬酸钠缓冲液中，在半小时内注射完)，1W后，禁食但不禁水12h，尾静脉取血，应用血糖仪测定空腹血糖值(FBG)，其中FBG≥7.8mmol/l者为2型糖尿病模型成功。试验动物在动物中心12小时明暗交替的标准化饲养间喂养，室温在20-25℃，室内相对的湿度45％-60％，饲养间内通风良好，试验期间给予标准颗粒的正常饲料和高脂高糖饲料，试验动物自由饮水。
1.2试验动物分组及给药
成功造模的大鼠选取50只，随机分为5组，分别是：2型糖尿病模型组、阳性药物对照组、蛋白高浓度组、蛋白中浓度组及蛋白低浓度组。阳性对照组药物为盐酸二甲双胍，浓度200mg/kg，蛋白给药组分别给予腹腔注射蛋白400ug/kg，300ug/kg，200ug/kg。正常对照组，模型组等体积腹腔注射纯净水，蛋白给药每周一次，二甲双胍每天一次，连续给药6W。试验期间给予标准颗粒的正常饲料，高脂高糖饲料，试验动物自由饮水。
2实验步骤
试验给药前，禁食但不禁水12h，血糖仪测空腹血糖值。试验结束时，禁食但不禁水12h，测空腹血糖值。腹腔注射葡萄糖在0、30、60、90、120min测血糖值，2天后禁食8h杀鼠，腹主动脉取血1.5ml，3000r/min x 5min x2，离心后取血清及下沉的红细胞。大鼠采取的血清主要用于生化指标的检测。
3结果
结果表1所示，RTB突变体干预后2型糖尿病大鼠的饮水量和体重较模型组明显改善；结果表2所示，给予RTB蛋白突变体干预后，空腹血糖(FBG)和糖化血红蛋白(GHb)水平明显改善；结果表3所示，RTB蛋白有较好的改善甘油三酯(TG)和总胆固醇(TCH)的效果；结果表4所示，糖耐量实验证明RTB有较高的改善2型糖尿病耐糖水平的效果。
表1各试验组试验前后饮水量和体重的比较(均数±标准差)
Figure PCTCN2018111237-appb-000001
注：与正常组比较， △P<0.05， △△P<0.01；与模型组比较， #P<0.05， ##P<0.01。
表2各试验组试验前后FBG水平与试验后GHb水平的比较(均数±标准差)
Figure PCTCN2018111237-appb-000002
注：与正常组比较， △P<0.05， △△P<0.01；与模型组比较， #P<0.05， ##P<0.01。
表3各组TG和TCH比较
Figure PCTCN2018111237-appb-000003
注：与模型组比较， #P<0.05， ##P<0.01。
表4各试验组试验前后糖耐量水平的比较(均数±标准差)
Figure PCTCN2018111237-appb-000004
注：与正常组比较， △P<0.05， △△P<0.01；与模型组比较， #P<0.05， ##P<0.01。
以上对本发明的一个实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。

Claims (10)

  1. 包含SEQ ID：1序列的蛋白在制备治疗2型糖尿病药物中的应用。
  2. 根据权利要求1所述的的蛋白，其特征在于：该蛋白可降低2型糖尿病模型的大鼠空腹血糖水平。
  3. 根据权利要求1所述的的蛋白，其特征在于：该蛋白可降低2型糖尿病模型的大鼠糖化血红蛋白水平。
  4. 根据权利要求1所述的的蛋白，其特征在于：该蛋白可降低2型糖尿病模型的大鼠的饮水量。
  5. 根据权利要求1所述的的蛋白，其特征在于：该蛋白可增加2型糖尿病模型的大鼠的体重，改善消瘦症状。
  6. 根据权利要求1所述的的蛋白，其特征在于：该蛋白可改善2型糖尿病模型的大鼠的甘油三酯(TG)和总胆固醇(TCH)水平。
  7. 根据权利要求1所述的的蛋白，其特征在于：该蛋白可改善2型糖尿病模型的大鼠的耐糖能力。
  8. 根据权利要求1所述的的蛋白，其特征在于：该蛋白通过大肠杆菌表达系统制备。
  9. 根据权利要求8所述的蛋白的制备方法，所述大肠杆菌表达系统所用载体为PET系列载体
  10. 根据权利要求8所述的蛋白的制备方法，所述大肠杆菌表达系统所用表达菌种为BL21。
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