CN107226850B - 一种抑制α糖苷酶活性的多肽及其应用 - Google Patents
一种抑制α糖苷酶活性的多肽及其应用 Download PDFInfo
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- CN107226850B CN107226850B CN201710373194.3A CN201710373194A CN107226850B CN 107226850 B CN107226850 B CN 107226850B CN 201710373194 A CN201710373194 A CN 201710373194A CN 107226850 B CN107226850 B CN 107226850B
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Abstract
本发明属于生物制药领域，具体属于一种抑制α糖苷酶活性的多肽及其应用，尤其涉及天然提取亚麻双功能多肽和原核重组表达多肽，以及它们在功能食品及降糖药物制备中的应用。这些多肽的氨基酸序列具体为：SEQ ID NO：1至SEQ ID NO：6。所述多肽可通过从天然亚麻中提取，人工合成、或通过原核细胞或真核细胞表达纯化获得。
Description
技术领域
本发明涉及生物制药领域，具体涉及一种可口服的抑制α糖苷酶活性的多肽及其衍生物制备和用途。
背景技术
糖尿病作为一种慢性代谢疾病，具有多种并发症，会对人体的神经系统、泌尿系统以及视觉系统等产生重大的影响。对于糖尿病的临床治疗来说，主要包括两个方面，一方面注射可以降低血糖的胰岛素。另一方面是口服降血糖药物，主要包括：胰岛素促泌剂、α-葡萄糖苷酶抑制剂、双胍类以及噻唑烷二酮类四种口服降糖药。α-葡萄糖苷酶抑制剂可以抑制小肠中糖类的吸收，延缓D-葡萄糖释放和吸收，以一种非侵害型的方式有效地调节了血糖水平和胰岛素反应。目前α糖苷酶抑制剂的药物多以阿卡波糖、米格列醇、伏格波列糖等有机小分子为主。现在有文献报道的具有α糖苷酶抑制活性的天然植物的产物集中在黄酮类、皂甙类、植物酸类以及多糖。只有豆科的白色扁豆、豇豆、大花田菁中有文献报道存在抑制α糖苷酶蛋白类活性成分。本发明中兼具抑制α糖苷酶活性和胰蛋白酶活性的多肽属于首次提及。
发明内容
本发明的目的是提供一种抑制α糖苷酶活性的多肽，所述多肽有的兼具抗胰蛋白酶活性能力。
本发明提供的一种抑制α糖苷酶活性的多肽(亚麻多肽，简称LUTI)，该多肽的制备，参见发明人公开专利“一种从亚麻蛋白粉中提取胰蛋白酶抑制剂方法”(公开号：CN105566491A)。本发明在对多肽的生物学活性测定中，发现该多肽不仅具有胰蛋白酶抑制活性，还具有α糖苷酶抑制活性，适合开发功能食品或口服降糖药物。本发明人委托公司对该多肽进行质谱分析，获得了多肽的氨基酸序列信息。所述亚麻多肽，具有序列表SEQ IDNO：6所示氨基酸序列。
本发明又根据LUTI的氨基酸序列序列表SEQ ID NO：6，首先进行密码子优化，化学合成适合大肠杆菌表达的编码基因，再经多点氨基酸添加、替代的突变，获得一些或提高α糖苷酶抑制活性，或提高胰蛋白酶抑制活性，或两者均提高的重组多肽，简称DFI，这些多肽具有序列表SEQ ID NO：1所示氨基酸序列，其可应用于功能食品、生物制药领域。
所述兼具胰蛋白酶抑制活性和α糖苷酶抑制活性重组多肽，具有序列表SEQ IDNO：2所示氨基酸序列。
所述兼具胰蛋白酶抑制活性和α糖苷酶抑制活性重组多肽，具有序列表SEQ IDNO：3所示氨基酸序列。
所述具有α糖苷酶抑制活性重组多肽，具有序列表SEQ ID NO：4所示氨基酸序列。
所述具有α糖苷酶抑制活性重组多肽，具有序列表SEQ ID NO：5所示氨基酸序列。
上述的任一抑制α糖苷酶活性的多肽可在制备降糖功能食品及口服降血糖药物中应用。
本发明的抑制α糖苷酶活性的多肽由于兼具胰蛋白酶抑制活性，具有较强的抗肠道蛋白酶水解能力因此可通过口服给药，在Ⅱ型糖尿病治疗中有很好的应用前景。
下面结合具体实施例和附图对本发明做进一步说明。
附图说明
图1.重组多肽DFI1的制备过程电泳图
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法。
天然提取或重组表达的多肽的简称代码与序列表中序列号的对应关系为：DFI1(SEQ ID NO：2)；DFI2(SEQ ID NO：3)；DFI3(SEQ ID NO：4)；DFI4(SEQ ID NO：5)；LUTI(SEQID NO：6)
实施例1.亚麻多肽氨基酸序列的获得
亚麻多肽(简称LUTI)的制备：在发明人公开了专利“一种从亚麻蛋白粉中提取胰蛋白酶抑制剂方法”(公开号：CN105566491A)后，对多肽的生物学活性测定中，发现该多肽不仅具有胰蛋白酶抑制活性，还具有α糖苷酶抑制活性。本发明人委托公司对该多肽进行质谱分析，获得了多肽的氨基酸序列信息，即具有序列表SEQ ID NO：6所示氨基酸序列。
实施例2.重组多肽的制备
根据LUTI的氨基酸序列，利用商业公司化学合成编码DFI1，DFI2,DFI3,DFI4多肽的核苷酸编码序列，然后利用5’端添加BamHI的正向引物(ATGGATCCAGCCGTCGTTGCCCG)，3’端添加EcoRI的反向引物(ATGAATTCGGTAATATGCGGCAC)，以合成基因为模板，进行PCR扩增。PCR反应条件：95℃5min，95℃30s，53℃30s，72℃40s，72℃10min，30个循环；利用限制性内切酶BamH I、EcoR I对PCR产物进行酶切，连接到经相同酶切割的表达载体质粒pGEX-6p-1，构建pGEX-6p-1-DFI1或DFI2或DFI3或DFI4的重组子，然后将重组子转入E.coli X10感受态细胞中，进行菌落PCR筛选，阳性克隆经测序分析确定。
0.5μl浓度为200ng/μl重组质粒pGEX-6p-1-DFI1或DFI2或DFI3或DFI4，迅速转入100μl冻融状态的E.coli Origami(DE3)感受态中，冰浴30分钟，42℃热击90秒，均匀涂布于LB固体平板培养基(含100μg/ml Amp)表面，37℃倒置过夜培养。次日挑取单菌落于5ml LB(含100μg/ml氨苄青霉素)液体培养基中，37℃、200rpm过夜震荡培养。然后将上述培养液按1％(v/v)的比例接种至500ml新鲜的LB液体培养基(含100μg/ml Amp)中扩大培养至OD600＝0.6～0.7时，加入IPTG使其终浓度达到0.3mM，16℃过夜诱导表达；8000rpm离心收集菌体。用30mL pH7.3磷酸缓冲液重悬菌体，冰上超声裂解菌体，13000r/min离心30min，收集上清，并上样至经pH7.3 PBS溶液平衡好的GST-Sepharose4B亲和层析纯化，然后GST-3C蛋白酶切割融合蛋白，再次利用GST-Sepharose4B纯化酶切产物，收集GST-Sepharose4B柱穿透样品，上样至缓冲液为pH7.3磷酸缓冲液平衡的Sephacryl S-200凝胶柱，收集目的蛋白，过滤除菌后保存于-80℃。
图1为重组多肽DFI1的制备过程电泳图，图中：1、表达GST-DFI1蛋白大肠杆菌BL21全菌样品；2、菌体超声破碎后离心上清样品；3、菌体超声破碎后离心沉淀样品；4、GST-Sepharose4B亲和柱穿透样品；5、GST-Sepharose4B亲和柱纯化样品；6、Sephacryl S-200凝胶柱纯化样品；7、GST-DFI1酶切后的混合样；8、重组的DFI1样品。
DFI2、DFI3和DFI4的制备过程电泳图同DFI1的基本相同。
实施例3.亚麻多肽以及重组多肽体外活性测定
α-葡萄糖苷酶抑制活性测定：以4-硝基苯-β-D-吡喃半乳糖苷(pNPG)为底物，取一定量的多肽(LUTI或DFI1或DFI2或DFI3或DFI4)溶液与37.5μL pNPG溶液(2×10-3mol/L)混合后，用0.2mol/L磷酸缓冲液中(pH6.8)补足体积至450μL，于37℃恒温水浴保温10min后，加入50μL来源于酵母的α-葡萄糖苷酶(0.5U/mL)，37℃反应10min，加入2.5mL 0.1mol/LNa2CO3溶液(pH 10.95)终止反应，在410nm处测其吸光值，以不加多肽试样为空白对照。
α-葡萄糖苷酶活力单位定义为：37℃下，pH为6.8的缓冲体系中，每分钟水解pNPG生成1μmol/L PNP所需要的酶量为一个活力单位。α-葡萄糖苷酶抑制剂活力单位定义为：在相同条件下，降低一个酶活性单位所需的抑制剂量。
抑制率(％)＝(△A空白组410－△A实验组410)/△A空白组410×100％
胰蛋白酶抑制活性的测定：依据胰蛋白酶能特异性酶解底物BAPNA,生成显色物质pNA,在410nm处有最大吸收峰，而胰蛋白酶抑制剂的加入可阻止胰蛋白酶对BAPNA的酶解，从而抑制反应后的吸光度值的增加。取一定量的多肽(LUTI或DFI1或DFI2或DFI3或DFI4)样品与0.4mL 0.2mg/mL的胰酶，在37℃下保温10min后，加入2mL 0.5mg/mL底物BAPNA，再于37℃下保温10min后，加入0.5mL醋酸溶液(w/v为33％)终止反应，在OD410下测量吸光度值。以不加抑制剂样品为空白对照。37℃下，每分钟水解BAPNA生成1μM对硝基苯胺PNA所需要的酶量为一个活力单位。在相同条件下，降低一个酶活性单位所需的抑制剂量，为一个抑制剂活性单位。
抑制率(％)＝(△A空白组410－△A实验组410)/△A空白组410×100％
多肽抑制α糖苷酶活性和胰蛋白酶活性的测定结果见表1。
表1亚麻胰蛋白酶抑制剂(LUTI)及重组双功能多肽(DFI)对α糖苷酶活性和胰蛋白酶抑制活性IC50值测定
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Gly Pro Leu Gly Ser Ser Arg Arg Ala Pro Gly Lys Asn Ala Trp Pro
1 5 10 15
Glu Leu Val Gly Lys Ser Gly Asn Met Ala Ala Ala Thr Val Glu Arg
20 25 30
Glu Asn Arg Asn Val His Ala Ile Val Leu Lys Glu Gly Ser Ala Met
35 40 45
Thr Lys Asp Phe Arg Cys Asp Arg Val Trp Val Ile Val Asn Asp His
50 55 60
Gly Val Val Thr Ser Val Pro His Ile Thr
65 70
<210> 4
<211> 74
<212> PRT
<213> Artificial Sequence
<220>
<223> 人工合成核苷酸序列，大肠杆菌重组表达
<220>
<221> DFI3
<222> (1)..(74)
<400> 4
Gly Pro Leu Gly Ser Ser Arg Arg Cys Pro Gly Lys Asn Ala Trp Pro
1 5 10 15
Glu Leu Val Gly Lys Ser Gly Asn Met Ala Ala Ala Thr Val Glu Arg
20 25 30
Glu Asn Arg Asn Val His Ala Ile Val Leu Lys Glu Gly Ser Ala Met
35 40 45
Thr Lys Asp Phe Arg Ala Asp Arg Val Trp Val Ile Val Asn Asp His
50 55 60
Gly Val Val Thr Ser Val Pro His Ile Thr
65 70
<210> 5
<211> 74
<212> PRT
<213> Artificial Sequence
<220>
<223> 人工合成核甘酸序列，大肠杆菌重组表达
<220>
<221> DFI4
<222> (1)..(74)
<400> 5
Gly Pro Leu Gly Ser Ser Arg Arg Ala Pro Gly Lys Asn Ala Trp Pro
1 5 10 15
Glu Leu Val Gly Lys Ser Gly Asn Met Ala Ala Ala Thr Val Glu Arg
20 25 30
Glu Asn Arg Asn Val His Ala Ile Val Leu Lys Glu Gly Ser Ala Met
35 40 45
Thr Lys Asp Phe Arg Ala Asp Arg Val Trp Val Ile Val Asn Asp His
50 55 60
Gly Val Val Thr Ser Val Pro His Ile Thr
65 70
<210> 6
<211> 69
<212> PRT
<213> Linum usitatissimum
<220>
<221> LUTI
<222> (1)..(69)
<400> 6
Ser Arg Arg Cys Pro Gly Lys Asn Ala Trp Pro Glu Leu Val Gly Lys
1 5 10 15
Ser Gly Asn Met Ala Ala Ala Thr Val Glu Arg Glu Asn Arg Asn Val
20 25 30
His Ala Ile Val Leu Lys Glu Gly Ser Ala Met Thr Lys Asp Phe Arg
35 40 45
Cys Asp Arg Val Trp Val Ile Val Asn Asp His Gly Val Val Thr Ser
50 55 60
Val Pro His Ile Thr
65
Claims (6)
1.一种抑制α糖苷酶活性的多肽，其特征是：所述多肽为序列表SEQ ID NO：2所示氨基酸序列。
2.一种抑制α糖苷酶活性的多肽，其特征是：所述多肽为序列表SEQ ID NO：3所示氨基酸序列。
3.一种抑制α糖苷酶活性的多肽，其特征是：所述多肽为序列表SEQ ID NO：4所示氨基酸序列。
4.一种抑制α糖苷酶活性的多肽，其特征是：所述多肽为序列表SEQ ID NO：5所示氨基酸序列。
5.权利要求1-4所述的任一抑制α糖苷酶活性的多肽在制备降糖功能食品中的应用。
6.权利要求1-4所述的任一抑制α糖苷酶活性的多肽在制备口服降血糖药物中的应用。
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| CN110317251B (zh) * | 2019-07-10 | 2022-12-30 | 山西大学 | 降糖多肽及其制备方法和应用 |
| CN113735942B (zh) * | 2021-09-22 | 2023-01-31 | 山西大学 | 重组降糖多肽及其制备方法与应用 |
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|---|---|---|---|---|
| CN105566491A (zh) * | 2016-03-08 | 2016-05-11 | 山西大学 | 一种从亚麻蛋白粉中提取胰蛋白酶抑制剂的方法 |
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| CN105566491A (zh) * | 2016-03-08 | 2016-05-11 | 山西大学 | 一种从亚麻蛋白粉中提取胰蛋白酶抑制剂的方法 |
Non-Patent Citations (4)
| Title |
|---|
| Determination of a High Precision Structure of a Novel;Tomasz Cierpicki等;《J. Mol. Biol》;20000105;第302卷;第1179-1182页 * |
| Irena Lorenc-Kubis等.Isolation and Amino Acid Sequence of a Serine Proteinase Inhibitor from Common Flax(Linum usitatissimum) Seeds.《CHEMBIOCHEM》.2001,(第2期),第45-51页. * |
| Isolation and Amino Acid Sequence of a Serine Proteinase Inhibitor from Common Flax(Linum usitatissimum) Seeds;Irena Lorenc-Kubis等;《CHEMBIOCHEM》;20010504(第2期);第48页图6 * |
| 亚麻种子中胰蛋白酶抑制剂的分离纯化及性质的研究;陈颖璐等;《食品工业科技》;20160809;第37卷(第22期);第238-239页结论部分 * |
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