CN113717269B - 一种重组的变体fgf21蛋白及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种重组人成纤维细胞生长因子21类似物(mFGF21)及其制备方法。将重组mFGF21蛋白编码基因与表达载体连接得到重组质粒后转化宿主细胞，筛选阳性稳定表达工程菌进行高密度发酵，离心收集菌体进行高压匀浆破碎，经包涵体富集、洗涤、变性、复性和缓冲液置换工序，并采用离子交换层析进行精纯和膜分离技术的后处理，获得电泳及液相纯度大于98％的mFGF21蛋白。本发明还公开了重组mFGF21蛋白在治疗肥胖、糖尿病、高脂血症和非酒精性脂肪性肝病等代谢疾病上的应用。相较于野生型hFGF21，本发明mFGF21蛋白的稳定性和生物学活性明显提高，且在改善模型动物的血糖血脂水平及脂肪肝上的效果更显著。
Description
技术领域
本发明涉及生物医药技术领域，具体讲是一种重组的变体FGF21蛋白及其制备方法和应用。
背景技术
随着生活水平的不断提高，以糖尿病(DM)和非酒精性脂肪肝病(NASH)为代表的代谢类疾病成为威胁人类健康的隐形杀手。我国胰岛素抵抗型糖尿病患者(T2DM)超过1亿人，发病后期会产生失明、神经病变、高血压、肝肾脏病变和心血管疾病等并发症，2025年全球市场达到1200亿美金。脂肪肝已经成为被严重低估的重大健康威胁，患病人数已超过糖尿病(T2DM 1亿)，脂肪肝除了会引发肝脏严重病变乃至癌变之外，还可能引发心血管及代谢疾病，或者加快发病进程。国内约有近4.3亿的人群患有非酒精性脂肪肝，全球患病率高达25％，超过17.5亿人，2025年全球市场将达到400亿美金。
DM疾病如果不能得到有效控制，可引起体内多系统损害，导致眼、肾、神经、心脏、血管等组织的慢性进行性病变，引起功能缺陷乃至衰竭。糖尿病因其并发症多、致残率高的特点已成为现阶段全世界需要着力应对的公共卫生问题。目前临床上尚无治愈的方法，患者需要终身服药以维持血糖平稳。同样，NASH疾病如不治疗后期造成肝纤维化和肝癌的风险将明显增加，而在该疾病的治疗方面，目前尚无相关药物批准上市。
成纤维细胞生长因子(FGF21)是成纤维细胞生长因子家族的一名新成员，其氨基酸序列与有FGF19亚家族成员之间具有较高的同源性。大量实验表明FGF21是体内又一个可以独立调节血糖和血脂的调节因子，体外能够促进3T3-L1脂肪细胞消耗葡萄糖，在体内具有降低血糖水平、降低甘油三酯、升高高密度胆固醇脂蛋白、降低低密度胆固醇脂蛋白等功能，并且不会产生低血糖、高胰岛素血症、体重增加及水肿等副作用。随着研究的进行，研究人员发现FGF-21不仅在糖脂代谢上具有重要的调节作用，其功能还涉及改善胰岛β细胞机能，延缓化学诱导的肝癌产生，降低体重、减少脂肪堆积等方面。这些特征赋予了FGF21成为治疗糖脂代谢异常的代谢综合征新药的必备条件，作为安全可靠的代谢调节因子FGF21很有潜力成为改善胰岛素抵抗的新型药物。故FGF21在开发成治疗糖尿病、肥胖、非酒精性脂肪肝等多种代谢疾病上具有重要的意义。但临床试验发现FGF21在糖尿病患者中并没有显著改善血糖的作用，这可能与设计的FGF21突变体的生物学活性和体内稳定性有关，所以对FGF21进行基因工程改造和化学修饰逐渐成为近年来研究的热点。
发明内容
鉴此，本发明通过计算机辅助预测及试验，设计并构建了变体mFGF21蛋白，通过优化表达及纯化工艺制备了具有稳定性和生物学活性较好的mFGF21蛋白。动物实验结果显示mFGF21蛋白在肥胖症、糖尿病及NASH模型小鼠上表现了良好的功效，且治疗效果显著优于野生型FGF21(hFGF21)。
本发明的目的是提供一种新的变体mFGF21蛋白，其氨基酸序列如SEQ ID NO:2所示；或与上述序列同源性高(例如至少95％、至少98％、至少99％同源性或具有一个或更多个氨基酸取代(例如保守性取代)、缺失和/或添加的序列)；或包含至少一个在第56位或第59位或第69位或第122位的氨基酸突变，其中第56位的氨基酸突变选自如下任意一个氨基酸突变：K56R；K56A；K56H；K56G；K56L；K56I，第59位的氨基酸突变选自如下任意一个：K59R；K59A；K59H；K59L；K59I，第69位的氨基酸突变选自如下任意一个氨基酸突变：K69R；K69A；K69H；K69L；K69I，第122位的氨基酸突变选自如下任意一个氨基酸突变：K122R；K122A；K122H；K122L；K122I。
本发明的另一目的是提供编码所述mFGF21蛋白的DNA序列及含该DNA序列的载体，还包括含该载体的宿主细胞。
进一步地，所述的变体mFGF21蛋白的制备方法，包括如下步骤：
步骤一：携带目的蛋白基因表达载体的构建：将合成的序列表中SEQ ID NO:1的基因片段与原核表达载体连接，转化大肠杆菌，获得表达菌种。
步骤二：mFGF21蛋白的表达：培养重组表达菌种并诱导其生产目标蛋白；
步骤三：mFGF21蛋白的纯化：收获经步骤二处理的表达菌种，经破碎、离心、膜分离、离子交换层析和超滤等工序，最终获得高纯度的目标蛋白。
进一步地，本发明还提供一种治疗代谢类疾病的含有上述mFGF21蛋白的药物组合物，可用于治疗糖尿病、肥胖症及非酒精性脂肪肝或相关疾病包括降低肝脏重量和肝脏甘油三酯的含量、修复肝脏损伤，改善非酒精性脂肪性肝炎、肝损伤、肝硬化等相关的代谢综合征。
再一方面，本发明还提供所述药物组合物在制备预防或治疗代谢类疾病药物中的用途。
进一步地，本发明还公开了一种修饰产物，采用药学上可接受的化学修饰物对mFGF21蛋白进行修饰而得，化学修饰物选自以下的一种或多种：聚合物(例如聚乙二醇(PEG)，羟乙基淀粉(HES)，透明质酸，聚唾液酸)，非结构化(多)肽链(例如PAS，XTEN)，类弹性蛋白样多肽(ELP)，血清蛋白(例如白蛋白)，血清蛋白结合分子(例如白蛋白结合域(ABD)，白蛋白结合脂肪酸)，抗体，免疫球蛋白，免疫球蛋白的Fc区/域和免疫球蛋白结合域。
本发明的变体FGF21蛋白的制备优选大肠杆菌表达系统，结合膜分离和色谱层析技术，发展了一种具有高表达、高产率、低成本的整体纯化工艺，可稳定高效制备高纯度的目标蛋白。
本发明的有益效果：
(1)本发明的变体FGF21蛋白相比野生型hFGF21具有更长效、更稳定、更好的治疗胖症，糖尿病，高血糖症，血脂异常，非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肝损伤、肝硬化、肝癌等代谢疾病的功效。
(2)本发明提供了一种高效稳定的FGF21蛋白的整体纯化方法。相对于真核表达系统(表达量约在30-70mg/L)，本发明采用大肠杆菌体系及适配的表达载体可获得较高的表达量，这样将显著降低了生产成本；并且集成膜分离和色谱分离技术开发的整体纯化工艺简便和收率高，该方法具有稳定、易于线性方法和高产量等优势，为后期蛋白药物的工业化研究奠定了坚实的基础。
附图说明
图1是本发明hFGF21和mFGF21蛋白在大肠杆菌中的表达鉴定分析图；
图2是本发明纯化mFGF21蛋白的电泳和高效液相色谱分析图；
图3是本发明mFGF21蛋白的血浆孵育稳定性及体内半衰期；
图4是本发明mFGF21蛋白体外细胞活性检测图；
图5是本发明mFGF21蛋白调节HFHC饲料诱导的NASH模型小鼠血糖水平的效果图；
图6是本发明mFGF21蛋白控制HFHC饲料诱导的NASH模型小鼠体重的作用图；
图7是本发明mFGF21蛋白改善HFHC饲料诱导的NASH模型小鼠血脂水平的作用图；
图8是本发明mFGF21蛋白改善HFHC饲料诱导的NASH模型小鼠肝损伤的作用图；
图9是本发明mFGF21蛋白对HFHC饲料诱导的NASH模型小鼠脂肪肝的治疗效果图；
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
说明：本发明中涉及的基因的设计、合成和克隆、表达载体的构建、核酸提取、测序和鉴定，以及表达产物的分离和纯化等操作步骤，可按照本领域已知的技术进行(参见CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1
FGF21蛋白的表达及纯化
(1)表达载体的构建
将设计好的mFGF21基因(见序列表SEQ ID NO:1)和野生型的hFGF21基因(GenBank:PA260867.1)由南京金斯瑞公司合成，并在目的基因两端添加Nde I与Hind III两酶切位点。将合成的含有酶切位点目的基因连接到pET30a(+)载体中，然后各自转化至大肠杆菌DH5α中。挑取阳性克隆，经过酶切鉴定并测序，即构建成功的重组表达载体pET30a-mFGF21和pET30a-hFGF21。
(2)FGF21蛋白的表达鉴定
将重组表达载体pET30a-mFGF21和pET30a-hFGF21分别转化BL21(DE3)感受态细胞中。挑取单菌落分别接种至20mL含Kan(50μg/mL)的LB培养基中，37℃培养12h，以体积比1:100接种于另一20mL含Kan(50μg/mL)的LB培养基中，37℃、180rpm培养，当A600在0.5左右时，加入IPTG(异丙基硫代半乳糖苷)至终浓度为0.5mmol/L继续培养，转速100rpm，37℃诱导4h后收获菌体。用PBS(磷酸缓冲盐溶液)重悬菌体，菌体破碎后离心，分别取上清和沉淀进行12％SDS-PAGE电泳分析。结果显示mFGF21和hFGF21蛋白在大肠杆菌中均以包涵体形式表达，而mFGF21蛋白的表达量较hFGF21蛋白要高(如图1所示)，mFGF21蛋白的表达量达约700mg/L，根据文献报道真核表达系统，如哺乳动物细胞系统表达量约50mg/L(中国生物化学与分子生物学学报，2012，28(8)：768-774)；毕赤酵母表达系统约30mg/L(MolecularMedicine Reports,2016；13:3619-3626.)；文献报道原核系统的产量约71mg/L(ApplMicrobiol Biotechnol.2019；103(2):719-730.)。而本专利大肠杆菌高密度发酵体系达产量达400mg/L。因此，可说明，采用大肠杆菌体系可获得较高的表达量。泳道M：蛋白标准分子量Marker；YQ：诱导前菌体；QJ：诱导后全菌；SQ：菌体破碎离心上清；CD：菌体破碎离心沉淀。
(3)FGF21蛋白的纯化
通过管式离心机收集高密度发酵表达菌体，并采用洗涤液(20mmol/L Tris，150mmol/L NaCl，pH8.0缓冲液)洗涤菌体3次，收获菌体中加入溶菌酶(1mg/mL)，4℃孵育30min，700-1000bar高压匀浆2次破碎菌体。继续采用管式离心机富集并洗涤包涵体(4倍pH8.0洗涤液洗滤)；采用pH 8.0含6M尿素变性液溶解包涵体后，40倍pH 8.0含1M尿素复性液稀释复性蛋白，最后通过10kD孔径膜包或中空纤维柱对复性液进行浓缩并缓冲液置换，从而得到复性好的目标蛋白。
将复性蛋白过两步Q阴离子交换层析柱纯化，通过增加盐离子浓度洗脱的方式纯化FGF21蛋白，最后采用10kD孔径膜包或中空纤维柱对纯化蛋白进行浓缩和缓冲液置换，获得目标FGF21蛋白。
采用SDS-PAGE电泳和高效液相色谱分析实施例1菌体表达和纯化制备的FGF21蛋白纯度和含量。结果显示纯化后变体mFGF21蛋白的电泳和液相纯度达98％以上，野生型hFGF21蛋白的液相纯度约97％(如图2所示)。经计算分析，mFGF21蛋白的整体纯化制备工艺产量(约400mg/L)，显著高于hFGF21蛋白(约80mg/L)。
对实施例1制备的mFGF21蛋白和hFGF21蛋白的体内外稳定性检测。具体方法和结果如下：
将mFGF21和hFGF21两种蛋白经0.22μm滤膜过滤除菌后，按照蛋白终浓度0.05mg/ml与小鼠血浆混合后37℃分别孵育12、24、36、48h后取样，通过ELISA双抗夹心法检测蛋白降解程度。mFGF21蛋白与血浆孵育48h后仍保留有89％的反应率，而野生型hFGF21仅有约21％的反应(如图3所示，##P<0.01,###P<0.001vs hFGF21)，说明相对于hFGF21蛋白，变体mFGF21蛋白具有良好的血浆稳定性。
选取体重约300g的SD大鼠10只，随机分为2组。每组分别皮下注射hFGF21和mFGF21，剂量1mg/kg，在给药后的0h、5min、15min、30min、45min、1h、2h、4h、8h、12h、24h采血约300μL，12000r/m离心10min，取上清进行蛋白含量检测。ELISA双抗夹心法测定蛋白的体内半衰期：用稀释好不同浓度的hFGF21和mFGF21蛋白(2μg/mL、0.2μg/mL、200ng/mL、20ng/mL和2ng/mL)分别建立蛋白浓度含量的标准曲线，应用ELISA测定各血清中目标蛋白的含量，统计学分析并计算出2种蛋白的体内半衰期。体内半衰期t1/2＝0.301*(t2-t1)/log(OD1/OD2)，其中OD1和OD2分别表示t1和t2时取出血清所对应酶标板上的平均光吸收值。结果如图3所示(##P<0.01vs hFGF21)，经公式计算出hFGF21和mFGF21的体内半衰期分别约为36min和155min，说明了相对于野生型hFGF21蛋白，改造后的变体mFGF21蛋白的体内半衰期显著提高。
对实施例1制备的mFGF21蛋白和hFGF21蛋白进行体外糖吸收活性检测，具体方法和测试结果如下：
HepG2细胞培养：HepG2细胞为人肝癌细胞株，培养条件为高糖DMEM培养基，其中加入10wt％新生牛血清NCS、青霉素100μg/mL、链霉素100μg/mL，在37℃、体积分数5％CO2、饱和湿度条件下培养。当细胞生长至高密度时，应进行传代，用0.25wt％胰蛋白酶液消化后，以体积比为1:3-1:5的比例将细胞接种于新的培养瓶内培养，取对数生长期的细胞用于实验。
HepG2细胞接种与处理：用质量浓度为0.25％胰蛋白酶消化液消化生长状态良好的HepG2细胞，离心收集细胞，按照2.5×104的密度将细胞接种于96孔板中继续培养，每孔中培养液体积为200μL。当细胞生长至均匀单层时，弃去上层培养液加入新鲜的无血清培养基继续培养12h。分别用不同浓度(10、100、1000nmol/L)的hFGF21和mFGF21蛋白刺激细胞24h。取培养基上清液用GOD-POD法检测培养基中的葡萄糖含量，每孔至少重复检测3次，37℃反应5-10min后，在500nm波长下测OD值。计算葡萄糖消耗率，并运用统计学分析实验结果。
培养液中葡萄糖浓度和细胞葡萄糖消耗率的计算公式如下：
葡萄糖浓度(mmol/L)＝OD样品/OD标准×5.55mmol/L
细胞葡萄糖消耗率(％)＝[(C空白葡萄糖-C给药葡萄糖)/C空白葡萄糖]×100％
结果如图4所示(#P<0.05,##P<0.01,###P<0.001vs hFGF21)，两种蛋白促细胞糖吸收率都呈现剂量依赖性，而mFGF21在低、中、高三种浓度下刺激细胞糖吸收率都显著高于hFGF21蛋白，说明了变体mFGF21蛋白的体外活性要明显优于hFGF21蛋白。
对实施例1制备的mFGF21蛋白和hFGF21蛋白在NASH模型小鼠上的药效评价，具体方法和结果如下：
实验动物及饲养：C57BL/6小鼠及动物实验均委托江苏集萃药康生物科技有限公司开展。
取SPF级6周龄雄性C57BL/6小鼠40只，喂食高脂高胆固醇高果糖饲料(HFHC)4个月后，剔除体重异常，筛选血糖及体重值相对接近均值的成模小鼠30只，随机分为模型对照组(Vehicle组)、hFGF21组、mFGF21组，每组10只。另取10只同周龄雄性C57BL/6小鼠作为正常对照组(Normal组)。于隔日早上8点半左右给予实验组相应的受试物一次，皮下注射，剂量2mg/kg；Vehicle和Normal组注射相同体积的生理盐水，连续给药4周。实验过程中自由饮食、饮水。期间监测小鼠饮食和体重状况。给药3天后，检测各实验组小鼠空腹血糖水平(6h)。给药4周后，各实验组小鼠处死(前夜禁食)，测定实验小鼠血清谷草转氨酶(AST)、谷丙转氨酶(ALT)、甘油三脂(TG)、总胆固醇(CHO)、高密度脂蛋白胆固醇(HDL)和低密度脂蛋白胆固醇(LDL)水平并进行肝脏病理组织切片染色和NASH指标检测。对所有实验数据进行统计学分析。
给药3d前后各实验组小鼠6h空腹血糖水平如图5所示(*P<0.05,**P<0.01vsVehicle；#P<0.05,##P<0.01vs hFGF21)，相对于模型对照组，mFGF21组小鼠的空腹血糖水平显著下降，并且mFGF21蛋白对血糖改善效果也显著优于hFGF21。说明了变体mFGF21蛋白在糖尿病疾病治疗上具有良好的潜力。
各实验组小鼠体重检测结果如图6所示(*P<0.05,**P<0.01vs Vehicle；#P<0.05,##P<0.01vs hFGF21)，相对于模型对照组，mFGF21和hFGF21蛋白均可显著降低小鼠体重，而mFGF21蛋白控制小鼠体重的能力较hFGF21要更明显，且停药后mFGF21小鼠体重回升较缓慢。说明了变体mFGF21蛋白在减肥方面的效果要显著优于hFGF21。
给药6周后，各实验组小鼠血清中血脂水平和肝功参数检测结果如图7和8所示(*P<0.05,**P<0.01vs Vehicle；#P<0.05,##P<0.01vs hFGF21)，相对于模型对照组，两种FGF21蛋白组小鼠血清中TG、CHO、LDL和AST、ALT水平均明显降低，HDL水平显著增加。而mFGF21蛋白改善模型小鼠血脂和肝功水平较hFGF21更显著。说明了mFGF21蛋白在治疗高脂血症和非酒精型脂肪肝疾病上将具有较好的效果。
脂肪肝改善方面，实验终点对各实验组小鼠肝组织切片进行HE染色和油红染色，并对组织切片进行评分(从脂肪变性、炎性病灶和气球样化三个方面打分)。判定标准为：总和＜3分，即为非NASH；评分总和＞5分，即为NASH；3分＜评分总和＜5分，即为非确定性NASH。病理切片评分结果显示(见图9，Bar＝50μm)，相对于模型对照组，hFGF21和mFGF21蛋白对模型小鼠的脂肪肝改善上都有明显的作用，根据NASH评分可知，mFGF21蛋白对于改善脂肪肝病变上具较好的治疗效果。
以上对实施例1的性能检测与结果显示，本发明设计并验证一种稳定性好和生物学活性高的变体FGF21蛋白，同时开发了高效的FGF21蛋白的纯化方法。通过模型动物药效评价试验发现变体FGF21蛋白针对NASH疾病上糖脂絮乱及脂肪肝症状的改善和治疗效果明显强于野生型hFGF21蛋白。综上说明了本发明的变体FGF21蛋白及其衍生物将在治疗代谢疾病方面具有良好的潜力及应用前景。
以上实施例描述了本发明的基本设计原理、主要特征及优点，本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。在不脱离本发明原理的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发明保护的范围内。
SEQUENCE LISTING
<110> 赣江中药创新中心
<120> 一种重组的变体FGF21蛋白及其制备方法和应用
<130> 赣江中药创新中心
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 543
<212> DNA
<213> SEQ ID NO:1
<400> 1
gctcctatac cagatagttc acccctatta caatttggtg gtcaagtgcg tcagcgctat 60
ctgtacaccg atgatgcaca gcaaaccgaa gcgcatctgg aaattcgtga ggacggcacg 120
gtgggtggtg cggcggatca gtctccggag agcctgctgc aactgcgtgc actggcgccg 180
ggcgtcatcc agattctggg tgttcatacc tcgcgcttct tatgccaacg cccagacggc 240
gctctgtacg gctccttgca cttcgacccg gaggcatgta gctttcgtga actgctgctt 300
gaggacggtt ataatgttta tcagagcgaa gcgcacggct tgccgttgca cttgccgggc 360
aaccgctccc cgcatcgtga tccggctccg cgtggtccgg cgagattcct gccgctgcct 420
ggtctgccgc cggcgctgcc ggagccgccg ggcatcctcg ccccacagcc gcccgacgtg 480
ggcagctccg atccgctcag catggttggt gccagccaag gtcgtagccc aagctacgaa 540
tct 543
<210> 2
<211> 181
<212> PRT
<213> SEQ ID NO:2
<400> 2
Ala Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Ala Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val His Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
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Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
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Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
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Gly Leu Pro Leu His Leu Pro Gly Asn Arg Ser Pro His Arg Asp Pro
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Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
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Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
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Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ala Ser Gln Gly Arg Ser
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Pro Ser Tyr Glu Ser
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Claims (7)
1.一种重组的变体mFGF21蛋白，其特征在于，其氨基酸序列：如SEQ ID NO: 2所示。
2.一种如权利要求1所述的变体mFGF21蛋白的编码基因，其特征在于，其核苷酸序列如序列表中SEQ ID NO:1所示。
3.携带如权利要求2所述编码基因的载体或宿主细胞。
4.一种如权利要求1所述的重组的变体mFGF21蛋白的制备方法，其特征在于，包括如下步骤：
步骤一：将合成的序列表中SEQ ID NO:1的基因片段与原核表达载体连接，转化大肠杆菌，获得表达菌种；
步骤二：培养重组表达菌种并诱导其生产目标蛋白；
步骤三：收获经步骤二处理的表达菌种，对其分离纯化工序，得到目标蛋白。
5.一种药物组合物，其特征在于，含有权利要求1所述mFGF21蛋白或采用权利要求4所述的制备方法制得的mFGF21蛋白。
6.根据权利要求5所述的一种药物组合物，其特征在于，还包括药学上可接受的载体、赋形剂或稀释剂。
7.一种如权利要求5或6的药物组合物在制备预防或治疗代谢疾病药物中的应用，其特征在于，所述代谢疾病包括糖尿病、肥胖症、高脂血症和非酒精性脂肪性肝病。
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