CN108794596B - 一种生物活性多肽enpraf及其制备方法和应用 - Google Patents
一种生物活性多肽enpraf及其制备方法和应用 Download PDFInfo
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- CN108794596B CN108794596B CN201810713330.3A CN201810713330A CN108794596B CN 108794596 B CN108794596 B CN 108794596B CN 201810713330 A CN201810713330 A CN 201810713330A CN 108794596 B CN108794596 B CN 108794596B
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- polypeptide
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Abstract
本发明涉及蛋白领域,具体涉及一种生物活性多肽ENPRAF及其制备方法和应用,生物活性多肽ENPRAF其氨基酸序列为Glu‑Asn‑Pro‑Arg‑Ala‑Phe。经过体外抗氧化实验、体内抗衰老实验,验证了多肽ENPRAF具有较好的抗氧化生物学活性和抗衰老活性,一方面,本发明的生物活性多肽ENPRAF具有较好的抗氧化活性,能够清除机体内的自由基,提高生命的质量;另一方面,能够提高体内抗过氧化酶系的活力,增强机体抵抗外源性刺激的功能,从而降低机体老化、衰老和生病的机率,对开发具有抗氧化功能、抗衰老功能的食品、保健品和药物具有十分重要的意义。
Description
技术领域
本发明涉及蛋白领域,尤其是涉及一种生物活性多肽ENPRAF及其制备方法和应用。
背景技术
在牛乳经乳酸菌发酵的过程中,牛乳中的一部分蛋白质被乳酸菌代谢利用,并发生了一系列生理生化反应,使蛋白质变为多肽或者游离的氨基酸,被人体消化吸收或通过小肠上皮细胞的吸收转运直接进入人体的血液循环。乳酸菌菌体也存在一些自身合成的蛋白质多肽片段,供细菌生长利用。在这些多肽中,有一部分具有特殊的生理功能,被称为“生物活性肽”。
在天然食物来源中寻找安全的生物活性肽尤为重要。近些年来,人们发现一些食物来源的多肽类物质具有良好的生物活性,如玉米短肽、大豆肽、牛乳多肽等。这些多肽可以通过微生物发酵、消化酶解等多种途径得到,并且大多具有生物活性的多肽是由2~20个氨基酸残基组成,分子量小于6000Da,含有一定量的疏水氨基酸、芳香族氨基酸。
氧化反应和氧化代谢对于食物和人体来说都是至关重要的,自由基和活性氧引起了一系列的氧化反应。当过量的自由基形成,它们会超过保护性酶如超氧化物歧化酶、过氧化氢酶的保护作用,从而导致脂质氧化、细胞凋亡等一系列的副作用产生。这一类的氧化反应,不仅影响含脂食物的保质期,也对人体的健康造成了一定的危害,如风湿性关节炎、糖尿病、动脉硬化等。此外,Collins等人2005年研究发现癌症的发生也与DNA的氧化损伤有关。
早期一些人工合成的抗氧化剂如丁基羟基茴香醚(BHA)、2,6-二叔丁基-4-甲基苯酚(BHT)被运用到食品中,作为脂质的抗氧化剂,但这些人工合成的添加剂对于人体都有潜在的风险。在天然抗氧化剂的研究过程中,来源于食物蛋白的抗氧化肽成为了热门研究之一。其不仅安全性高,比蛋白质等大分子营养物质更容易被吸收利用,能促进如钙、铁等微量营养素的吸收,还具有较好的抗氧化活性,应用前景广阔。
衰老是一个自然现象,且过程常伴有抗氧化水平、器官组织、免疫因子的变化,其中细胞因子发生着复杂的变化,如促炎细胞因子IL-6、IL-4、TNF-α等呈现增长的趋势,IL-6与TNF-a均被认为在老年性疾病的发生过程中扮演重要角色。随着遗传学和分子生物学的发展,生物衰老机理的研究取得了可喜的进展。研究人员通过利用一些模式生物,如小鼠、果蝇和秀丽线虫等的单一基因突变实验,发现有些基因能够显著增加这些生物体的寿命达6倍之多。
抗衰老肽作为一种新兴的抗衰老剂,在生理功能方面具有氨基酸所不能比拟的优势,其能对生物体内的酶产生促进或抑制作用,改善对矿物质及其他营养元素的吸收和利用,清除体内自由基,增强机体自身的抗氧化力,以延缓衰老。因此,生物活性肽的营养保健作用已成为国内外学者课题研究的重点。邱隽等人经实验研究发现,乳源性生物活性小肽能有效延长果蝇寿命,延缓其衰老,并且还具有较好的抗氧化作用,推测可能是其中富含琉基肽类。周之辉等发现牛初乳提取物能显著性提高老年人体内血清中SOD活力,减少其脂质过氧化物和增强机体抗氧化力,具有一定的抗衰老功能。
目前关于生物活性多肽的研究有很多,比如中国专利CN105254738A公布了一种来源于β-酪蛋白的乳源性生物活性多肽DELQDKIH,中国专利CN105254739A公布了一种来源于αs1-酪蛋白的乳源性生物活性多肽GTQYTD,中国专利CN105254740A公布了一种来源于αs2-酪蛋白的乳源性生物活性多肽NQFYQKF。
发明内容
本发明的目的在于提供一种生物活性多肽ENPRAF及其制备方法和应用。
本发明的目的可以通过以下技术方案来实现:
本发明第一方面,提供一种生物活性多肽ENPRAF,其氨基酸序列为Glu-Asn-Pro-Arg-Ala-Phe,如SEQ ID NO:1所示。
较优的,所述生物活性多肽来源于瑞士乳杆菌菌体蛋白。具体来源于>LBH_1022|m.953 LBH_1022|g.953 ORF LBH_1022|g.953 LBH_1022|m.953 type:complete len:565(+)LBH_1022:1-1695(+)蛋白,并且为此蛋白第229~234位的氨基酸残基。>LBH_1022|m.953 LBH_1022|g.953 ORF LBH_1022|g.953 LBH_1022|m.953 type:complete len:565(+)LBH_1022:1-1695(+)蛋白氨基酸序列如SEQ ID NO:3所示。
>LBH_1022|m.953 LBH_1022|g.953 ORF LBH_1022|g.953 LBH_1022|m.953type:complete len:565(+)LBH_1022:1-1695(+)蛋白的氨基酸序列以及对应的核苷酸序列为既有技术,编码此蛋白第229~234位氨基酸残基的核苷酸片段能编码成熟的生物活性多肽ENPRAF。
较优的,所述生物活性多肽具有抗氧化功能和抗衰老功能。
本发明第二方面,提供了编码所述生物活性多肽ENPRAF的核苷酸片段,其序列为:5’-aaa atc ctc gcg cat ttg-3’,如SEQ ID NO:2所示。
本发明第三方面,提供了所述生物活性多肽ENPRAF的制备方法,可以通过基因工程的方法人工合成,可以从瑞士乳杆菌菌体通过细胞破碎分离纯化的方法直接获得,可以直接通过化学合成制备。
本发明第四方面,提供了所述生物活性多肽ENPRAF在制备具有抗氧化功能的食品、保健品、药物或化妆品中的应用。
本发明第五方面,提供了所述生物活性多肽ENPRAF在制备具有抗衰老功能的食品、保健品或药物中的应用。
本发明第六方面,提供了所述生物活性多肽ENPRAF在制备同时具有抗氧化功能和抗衰老功能的食品、保健品或药物中的应用。
具体而言,本发明的生物活性多肽ENPRAF可以用于制备减少自由基对皮肤伤害的化妆品、制备具有抗氧化和/或抗衰老的药物;并且由于本发明的生物活性多肽ENPRAF通过胃肠道降解后的产物仍旧具有生物活性,因此还可以用于制备酸奶等食品、抗氧化的保健品,以及口服的用于制备具有抗氧化和/或抗衰老的药物。
本发明第七方面,提供了一种抗氧化产品,包括所述生物活性多肽ENPRAF或所述生物活性多肽ENPRAF的衍生物;所述的抗氧化产品包括抗氧化食品、抗氧化保健品、抗氧化药物或抗氧化化妆品;所述生物活性多肽ENPRAF的衍生物,是指在生物活性多肽ENPRAF的氨基酸侧链基团上、氨基端或羧基端进行羟基化、羧基化、羰基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰,得到的多肽衍生物。
本发明第八方面,提供了一种抗衰老产品,包括所述生物活性多肽ENPRAF或所述生物活性多肽ENPRAF的衍生物;所述的抗衰老产品包括抗衰老食品、抗衰老保健品或抗衰老药物;所述生物活性多肽ENPRAF的衍生物,是指在生物活性多肽ENPRAF的氨基酸侧链基团上、氨基端或羧基端进行羟基化、羧基化、羰基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰,得到的多肽衍生物。
本发明第九方面,提供了一种同时具有抗氧化功能和抗衰老功能的产品,包括所述生物活性多肽ENPRAF或所述生物活性多肽ENPRAF的衍生物;具有抗氧化功能和抗衰老功能的产品包括食品、保健品或药物;所述生物活性多肽ENPRAF的衍生物,是指在生物活性多肽ENPRAF的氨基酸侧链基团上、氨基端或羧基端进行羟基化、羧基化、羰基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰,得到的多肽衍生物。
本发明生物活性多肽ENPRAF的有益效果为:本发明的生物活性多肽ENPRAF具有较好的抗氧化活性和抗衰老活性;一方面,本发明的生物活性多肽ENPRAF具有较好的抗氧化活性,能够清除机体内的自由基,提高生命的质量;另一方面,能够提高体内抗过氧化酶系的活力,增强机体抵抗外源性刺激的功能,从而降低机体老化、衰老和生病的机率,对开发具有抗氧化功能、抗衰老功能的食品、保健品和药物具有十分重要的意义。
附图说明
图1:质量色谱提取图(m/z=734.3551);
图2:质荷比为734.3551的片段的二级质谱图;
图3:质荷比为734.3551的多肽az、by断裂情况;
图4:[DPPH·]甲醇标准曲线;
图5:生育酚Trolox标准曲线;
图6:不同浓度ENPRAF对秀丽线虫生殖能力的影响;
图7:不同培养条件下线虫在L4期时生长状态;
图8:生物活性多肽ENPRAF对秀丽线虫体长的影响;
图9:生物活性多肽ENPRAF对秀丽线虫寿命的影响。
具体实施方式
在进一步描述本发明具体实施方案之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明,具体可参见Sambrook等MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring HarborLaboratory Press,1989and Third edition,2001;Ausubel等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS INENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),AcademicPress,San Diego,1999;和METHODS IN MOLECULAR BIOLOGY,Vol.119,ChromatinProtocols(P.B.Becker,ed.)Humana Press,Totowa,1999等。
下面结合附图和具体实施例对本发明进行详细说明。
实施例1活性肽ENPRAF的人工合成
一、生物活性肽的合成
1.称取RINK树脂3g(取代度0.3mmol/g)于150ml的反应器中,用50ml的二氯甲烷(DCM)浸泡。
2.2小时后,用3倍树脂体积的氮-二甲基甲酰胺(DMF)洗涤树脂,然后抽干,如此重复四次,将树脂抽干后待用。
3.向反应器中加入一定量的20%哌啶(哌啶/DMF=1:4,v:v),放在脱色摇床上摇晃20min,以此来脱去树脂上的Fmoc保护基团。脱完保护后用3倍树脂体积的DMF洗涤四次,然后抽干。
4.取少量树脂用茚三酮(九井水合茚三酮)法检测(检A、检B各两滴,100℃反应1min),树脂有颜色,说明脱保护成功。
5.称取氨基酸Glu适量和1-羟基-苯骈三唑(HOBT)适量于50ml的离心管中,加入20ml的DMF将其溶解,然后加入3ml的N,N二异丙基碳二亚胺(DIC)振荡摇匀1min,待溶液澄清后加入到反应器中,然后将反应器置于30℃的摇床中反应。
6.2小时后,用一定量的醋酸酐封头(醋酸酐:DIEA:DCM=1:1:2,v:v:v)半小时,然后用3倍树脂体积的DMF洗涤四次,抽干待用。
7.向反应器中加入一定量的20%哌啶(哌啶/DMF=1:4,v:v),放在脱色摇床上摇晃20min,以此来脱去树脂上的Fmoc保护基团。脱完保护后用DMF洗涤四次,然后抽干。
8.取少量树脂用茚三酮(九井水合茚三酮)法检测(检A、检B各两滴,100℃反应1min),树脂有颜色,说明脱保护成功。
9.称取后面第二个氨基酸适量和HOBT适量于50ml的离心管中,加入25ml的DMF将其溶解,然后加入2.5ml的DIC振荡摇匀1min,待溶液澄清后加入到反应器中,然后将反应器置于30℃的摇床中反应。
10.1小时后,取少量树脂检测,用茚三酮法检测(检A、检B各两滴,100℃反应1min),若树脂为无色,说明反应完全;若树脂有颜色,说明缩合不完全,继续反应。
11.待反应完全后,用DMF洗涤树脂四次,然后抽干,向反应器中加入一定量的20%哌啶(哌啶/DMF=1:4,v:v),放在脱色摇床上摇晃20min,以此来脱去树脂上的Fmoc保护基团。脱完保护后用DMF洗涤四次,然后抽干检测保护是否脱去。
12.按照步骤9-11依次接上氨基酸Asn、Pro、Arg、Ala和Phe。
13.待接上最后一个氨基酸后,脱去保护,用DMF洗涤四次,然后用甲醇将树脂抽干。然后用95切割液(三氟乙酸:1,2乙二硫醇:3,异丙基硅烷:水=95:2:2:1,v:v:v)将多肽从树脂上切割下来(每克树脂加10ml切割液),并用冰乙醚(切割液:乙醚=1:9,v:v)离心沉降四次。
至此,人工合成了生物活性肽ENPRAF。
二、生物活性肽的确认
1)UPLC分析
UPLC条件如下:
仪器:Waters ACQUITY UPLC超高效液相-电喷雾-四级杆-飞行时间质谱仪
色谱柱规格:BEH C18色谱柱
流速:0.4mL/min
温度:50℃
紫外检测波长:210nm
进样量:2μL
梯度条件:A液:含有0.1%甲酸(v/v)的水,B液:含有0.1%甲酸(v/v)的乙腈
2)质谱分析
质谱条件如下:
离子方式:ES+
质量范围(m/z):100-1000
毛细管电压(Capillary)(kV):3.0
采样锥(V):35.0
离子源温度(℃):115
去溶剂温度(℃):350
去溶剂气流(L/hr):700.0
碰撞能量(eV):4.0
扫描时间(sec):0.25
内扫描时间(sec):0.02
根据以上分析方法,利用超高效液相-电喷雾-四级杆-飞行时间质谱,对生物活性肽ENPRAF进行色谱分析和质谱分析,其质量色谱提取图如图1所示,提取此峰的二级质谱图和az、by断裂情况如图2和3所示,可得此峰的多肽质荷比为734.3551Da,保留时间是31.6min。
3)结果
由图3可知,根据az、by断裂的情况,经过Mascot软件分析计算,得到质荷比734.3551Da的片段序列为Glu-Asn-Pro-Arg-Ala-Phe(ENPRAF),记为SEQ ID NO:1。该片段与LBH_1022|m.953 LBH_1022|g.953 ORF LBH_1022|g.953 LBH_1022|m.953 type:complete len:565(+)LBH_1022:1-1695(+)蛋白第229~234位的残基序列相对应,序列见SEQ ID NO:3。
实施例2生物活性肽的抗氧化活性实验
一、[DPPH·]法测定生物活性肽ENPRAF的体外抗氧化活性
1.实验试剂及仪器:
试剂:1,1-二苯基-2-三硝基苯肼(1,1-Diphenyl-2-picrylhydrazyl[DPPH·]),日本Wako公司生产;甲醇,上海国药公司提供;实施例1获得的生物活性多肽ENPRAF。
主要仪器:Sunrise酶标仪,奥地利Tecan公司产品;96孔细胞培养板,美国Millipore公司制造;分析天平,Meitelei-tolido公司产品。
2.实验方法:
(1)1mmol/L[DPPH·]甲醇溶液
用分析天平称取0.349mg[DPPH·]溶于1mL甲醇溶液中,配制得到的1mmol/L[DPPH·]甲醇溶液,锡纸避光保存,即配即用。
(2)[DPPH·]甲醇标准曲线的测定
在96孔板中按表1分别加入100μL[DPPH·]甲醇标准曲线样品,室温静置90min,用酶标仪在517nm处检测吸光值。
表1[DPPH·]甲醇标准曲线溶液配制
根据实验结果,使用Excel拟合曲线并计算回归方程,结果见图4(回归方程:y=-0.192x+0.2271,R2=0.9991)。[DPPH·]甲醇标准曲线的线性关系良好,相关系数为0.999,表明[DPPH·]甲醇标准曲线精密度和准确度均符合检测要求。从结果看,吸光度值与[DPPH·]含量呈反比关系,[DPPH·]含量越少,吸光值越高,即样品清除自由基的能力越强。
(3)[DPPH·]法测定生物活性肽ENPRAF的抗氧化活性
1)样品组:在96孔板中加入80μL浓度为1mmol/L[DPPH·]甲醇溶液、按表2分别加入20μL不同浓度的待测样品(ENPRAF)、阳性对照1(2.5mg/mL的Trolox)、阳性对照2(0.025mg/mL的Trolox),和阴性对照(植酸);
2)空白组:在同一96孔板上,以加入80μL浓度为1mmol/L[DPPH·]甲醇溶液和20μL去离子水的样品做空白对照。
待检测样品加样完毕后,室温静置90min,用酶标仪在517nm处检测吸光值。按照下式计算自由基清除率,实验结果见表2。
表2[DPPH·]法测定生物活性多肽的抗氧化活性结果
从表2可以看出,作为阳性对照的2.5mg/mL的Trolox在相同条件下具有最强的清除自由基的能力,几乎能清除溶液中所有的自由基,其次为0.025mg/mL的Trolox、植酸、活性多肽。多肽ENPRAF清除[DPPH·]自由基率随浓度变化呈现倒钟型,在浓度为2.5mg/mL处达到最高值,为26.67%。
二、ABTS法测定生物活性肽ENPRAF体外抗氧化能力
1.实验试剂和仪器:
总抗氧化能力检测试剂盒(Total Antioxidant Capacity Assay Kit with ABTS法),购自上海碧云天生物科技公司;ABTS溶液,氧化剂溶液,水溶性维生素E(Trolox溶液)(10mmol/L),实施例1获得的生物活性多肽ENPRAF。
主要仪器:Sunrise酶标仪,奥地利Tecan公司产品;96孔细胞培养板,美国Millipore公司制造;分析天平,Meitelei-tolido公司产品。
2.实验方法:
(1)ABTS工作液的配制
根据总抗氧化能力检测试剂盒说明书,将ABTS溶液和ABTS氧化剂溶液1:1混合,避光存放12-16h后使用。配好的ABTS母液在室温下避光存放,2-3天内稳定。使用前,用PBS稀释ABTS工作母液38-42倍,使得ABTS工作液的吸光度减去相应PBS空白对照后,A734为0.7±0.05,ABTS工作液锡纸避光保存,现配现用。
(2)生育酚(Trolox)标准曲线曲线的制作测定
在96孔板的每个检测孔中加入200μL ABTS工作液,标准曲线检测孔中按表3要求加入10μL用PBS稀释的生育酚(Trolox)溶液,空白对照孔中加入10μL PBS,轻轻混匀。室温孵育4min后,在734nm处检测吸光值。
表3生育酚(Trolox)标准曲线测定的溶液配制
根据实验的结果,用Excel拟合回归曲线并得出回归方程,结果见图5所示。Trolox标准曲线线性关系良好,其相关系数达到0.998,表明该标准曲线的准确度和精密性都符合检测要求,可用于后续结果计算。从图中可以看出,Trolox标准曲线与吸光值呈现良好的反比关系,Trolox溶液的浓度越高,其在734nm下吸光值越低,即所测样品的清除自由基能力越强。
(3)ABTS法测定生物活性多肽ENPRAF的抗氧化能力
在96孔板的每个检测孔中加入200μL ABTS工作液,样品检测孔中加入10μL待测样品,空白对照孔中加入10μL PBS,轻轻混匀。室温孵育4min后,用酶标仪在734nm处检测吸光值。根据标准曲线计算出样品的总抗氧化能力。总抗氧化能力表示方式以Trolox标准溶液的浓度来表示,按照下式计算自由基清除率,实验结果见表4。
总抗氧化能力(mmol/g)=CTrolox/CS
式中:CTrolox——与样品吸光值相同的Trolox标准溶液浓度(mmol/L)
CS——合成多肽样品的浓度(mg/mL)
表4 ABTS法测定生物活性多肽ENPRAF的总抗氧化能力结果
通过总抗氧化能力法(Total Antioxidant Capacity Assay Kit with ABTS法)对多肽ENPRAF的体外总抗氧化活性进行了测定,发现生物活性多肽ENPRAF相比空白组其吸光值有一定程度的降低,具有较好的还原氧化物质的能力。由表4可知,发现多肽ENPRAF的总抗氧化能力随着多肽浓度的升高而升高,在浓度为5mg/mL情况下,多肽ENPRAF的总抗氧化水平达到0.1849mmol/g,即在5mg/m L的浓度下,其总抗氧化能力与1mmol/L Trolox的总抗氧化能力相持平。因此,可认定发明的生物活性多肽ENPRAF具有显著的抗氧化能力。
实施例3生物活性肽的抗衰老活性实验
一、生物活性多肽ENPRAF对秀丽线虫生殖能力影响的实验
1.实验试剂及仪器:
试剂:秀丽线虫(Caenorhabditis elegans),复旦大学附属中西结合研究院;大肠菌株E.coli OP50,复旦大学附属中西结合研究院;琼脂粉,国药集团化学试剂有限公司;酵母粉,国药集团化学试剂有限公司;实施例1获得的生物活性多肽ENPRAF。
仪器设备:力康RO15纯水系统,力康生物医疗科技有限公司;G136T型Zealway智能高温灭菌锅,厦门致微仪器科技有限公司;THZ-32型台式恒温振荡器,上海精密实验设备有限公司;TDL-40B离心机,上海安亭科学仪器厂;卢湘仪GL-22M高速冷冻离心机,上海卢湘仪仪器有限公司;博迅BJ-CD SERIES生物安全柜,上海博讯实业有限公司;Nikko倒置电子显微镜,尼康株式会社。
2.实验方法:
(1)NGM平板制备
取大肠杆菌菌种于LB平板划线,挑取单菌落于10ml LB液体培养基中,37℃,200rpm,振荡培养24h,至OD600=0.4用于接种NGM平板喂养线虫。取100μL菌液涂于60mmNGM平板,注意菌液边缘应距离平板边缘0.5cm左右。已涂布的NGM平板在室温(21-25℃)过夜后即可使用。
(2)线虫培养
本实验中所用的线虫均为雌雄同体,在标准培养条件(温度20℃,湿度40%~60%)下培养生长。
(3)线虫的同期化处理
1)高氯酸钠漂白法
准备孕虫生长板(即板中80%以上虫子处于生殖期)2-3板,取5ml M9缓冲液冲洗2次,将缓冲液吸入15ml离心管中,1000r/min离心3min,弃去上清。加入5ml新配同期化漂白液,室温下剧烈振荡2.5min,以将成虫虫体腐蚀。离心,弃去上清。保证总处理时间不能超过5min,防止损伤虫卵。再加入M9缓冲液将沉淀重悬,混匀后离心,弃去上清,重复此过程3次。
2)限时产卵法
挑取若干条处于产卵期的线虫于同一平板内,挑取的具体数量以所需同期化线虫数目为依据。一般条件下,一条产卵期线虫能在1h以内产卵6个左右。在平板中培养0.5h后,挑出平板中线虫,则平板中的卵处于同一生长时期。
(4)指标测定
本实验以秀丽线虫作为动物模型,挑取同期化处理后的L4期线虫到相应浓度的NGM板中。每个浓度至少8条线虫,每个NGM板转入一条,记为0天,以后每天移至新板中直至线虫生殖基本不再产卵,在其进入产卵期之前对线虫总产卵数进行计数。
3.实验结果及分析:
实验结果如图6所示,与不喂食多肽ENPRAF的空白组相比较,喂食不同质量浓度的实验组中,其平均产卵数均有不同程度的增加。喂食的多肽ENPRAF浓度为300mg/L时,线虫平均产卵数与空白组相比具有极其显著的差异(P<0.01),而在其浓度为400mg/L、500mg/L时,与空白组相比却只呈现显著性差异(P<0.05),这也进一步证明了300mg/L为混合肽多肽ENPRAF作用最佳浓度,随着肽浓度的提高并不会抑制线虫生殖,而是其作用效果减弱。综上所述,多肽ENPRAF在一定浓度下能明显提升线虫的生殖能力。同时,此实验结果表明,多肽ENPRAF300mg/L为最适浓度。但随着浓度的增加,线虫的生殖能力却不再出现明显提高。
二、生物活性多肽ENPRAF对秀丽线虫体长影响的实验
1.实验试剂及仪器:
试剂:秀丽线虫(Caenorhabditis elegans),复旦大学附属中西结合研究院;大肠菌株E.coli OP50,复旦大学附属中西结合研究院;琼脂粉,国药集团化学试剂有限公司;酵母粉,国药集团化学试剂有限公司;实施例1获得的生物活性多肽ENPRAF。
仪器设备:力康RO15纯水系统,力康生物医疗科技有限公司;G136T型Zealway智能高温灭菌锅,厦门致微仪器科技有限公司;THZ-32型台式恒温振荡器,上海精密实验设备有限公司;TDL-40B离心机,上海安亭科学仪器厂;卢湘仪GL-22M高速冷冻离心机,上海卢湘仪仪器有限公司;博迅BJ-CD SERIES生物安全柜,上海博讯实业有限公司;Nikko倒置电子显微镜,尼康株式会社。
2.实验方法:
(1)NGM平板制备
取大肠杆菌菌种于LB平板划线,挑取单菌落于10ml LB液体培养基中,37℃,200rpm,振荡培养24h,至OD600=0.4用于接种NGM平板喂养线虫。取100μL菌液涂于60mmNGM平板,注意菌液边缘应距离平板边缘0.5cm左右。已涂布的NGM平板在室温(21-25℃)过夜后即可使用。
(2)线虫培养
本实验中所用的线虫均为雌雄同体,在标准培养条件(温度20℃,湿度40%~60%)下培养生长。
(3)线虫的同期化处理
1)高氯酸钠漂白法
准备孕虫生长板(即板中80%以上虫子处于生殖期)2-3板,取5ml M9缓冲液冲洗2次,将缓冲液吸入15ml离心管中,1000r/min离心3min,弃去上清。加入5ml新配同期化漂白液,室温下剧烈振荡2.5min,以将成虫虫体腐蚀。离心,弃去上清。保证总处理时间不能超过5min,防止损伤虫卵。再加入M9缓冲液将沉淀重悬,混匀后离心,弃去上清,重复此过程3次。
2)限时产卵法
挑取若干条处于产卵期的线虫于同一平板内,挑取的具体数量以所需同期化线虫数目为依据。一般条件下,一条产卵期线虫能在1h以内产卵6个左右。在平板中培养0.5h后,挑出平板中线虫,则平板中的卵处于同一生长时期。
(4)指标测定
实验分组:空白组和多肽组。不同组线虫,在同一时期下体长之间的差异,可以在一定程度上反映出该活性物质对于线虫生长发育的影响。各组同期化培养的线虫在生长至L2期(培养2天左右)时,分别挑取40条线虫至各自NGM平板,连续2天、3天、4天、5天、6天、8天、10天用倒置显微镜观察其生长状态,测定和记录其体长,每组取其平均值。
3.实验结果及分析:
在20℃的培养条件下,从线虫生长的L2期(第2天)开始观察,L3期(第3天)、L4期(第4天)、成虫期(第6天),连续观察8天,直至线虫生长的第10天,测定每个时间点下线虫的体长。结合图7和图8可以看出,各组线虫在L4期时其体长均为1000μm左右,无明显区别。同时,从线虫体长变化曲线中也可看出,实验组体长变化曲线也几乎和空白组体长变化曲线相重合,在线虫L3期(第3天)时,虽然线虫平均体长稍有区别,但是在统计学上并不呈现显著性差异。实验表明,多肽ENPRAF的浓度并不会影响线虫的生长。同时,也可发现线虫在L3期(第3天)至L4期(第4天),是其生长最为快速的阶段。
三、生物活性多肽ENPRAF对秀丽线虫寿命影响的实验
1.实验试剂及仪器:
试剂:秀丽线虫(Caenorhabditis elegans),复旦大学附属中西结合研究院;大肠菌株E.coli OP50,复旦大学附属中西结合研究院;琼脂粉,国药集团化学试剂有限公司;酵母粉,国药集团化学试剂有限公司;5-氟脲嘧啶,美国Sigma公司;实施例1获得的生物活性多肽ENPRAF。
仪器设备:力康RO15纯水系统,力康生物医疗科技有限公司;G136T型Zealway智能高温灭菌锅,厦门致微仪器科技有限公司;THZ-32型台式恒温振荡器,上海精密实验设备有限公司;TDL-40B离心机,上海安亭科学仪器厂;卢湘仪GL-22M高速冷冻离心机,上海卢湘仪仪器有限公司;博迅BJ-CD SERIES生物安全柜,上海博讯实业有限公司;Nikko倒置电子显微镜,尼康株式会社。
2.实验方法:
(1)NGM平板制备
取大肠杆菌菌种于LB平板划线,挑取单菌落于10ml LB液体培养基中,37℃,200rpm,振荡培养24h,至OD600=0.4用于接种NGM平板喂养线虫。取100μL菌液涂于60mmNGM平板,注意菌液边缘应距离平板边缘0.5cm左右。已涂布的NGM平板在室温(21-25℃)过夜后即可使用。
(2)线虫培养
本实验中所用的线虫均为雌雄同体,在标准培养条件(温度20℃,湿度40%~60%)下培养生长。
(3)线虫的同期化处理
1)高氯酸钠漂白法
准备孕虫生长板(即板中80%以上虫子处于生殖期)2-3板,取5ml M9缓冲液冲洗2次,将缓冲液吸入15ml离心管中,1000r/min离心3min,弃去上清。加入5ml新配同期化漂白液,室温下剧烈振荡2.5min,以将成虫虫体腐蚀。离心,弃去上清。保证总处理时间不能超过5min,防止损伤虫卵。再加入M9缓冲液将沉淀重悬,混匀后离心,弃去上清,重复此过程3次。
2)限时产卵法
挑取若干条处于产卵期的线虫于同一平板内,挑取的具体数量以所需同期化线虫数目为依据。一般条件下,一条产卵期线虫能在1h以内产卵6个左右。在平板中培养0.5h后,挑出平板中线虫,则平板中的卵处于同一生长时期。
(4)指标测定
实验分组:空白组和多肽组。选取L4期线虫若干条,同期化处理后,分别置于相应NGM板中;每组线虫数量不少于60条,此时记为0天,每天将它们转移到新板中,到生殖后期不再转移。每天记录线虫死亡及剔除出实验的条数。其中寿命实验每个NGM板中含有12.5mg/L 5-氟脲嘧啶以抑制线虫生殖。线虫死亡判断标准:无移动及吞咽动作,轻触后仍无任何反应。剔除标准:①逃离至平板壁或盖上而干死;②虫卵在体内孵化而成袋样虫:③钻入琼脂中。
3.实验结果及分析:
表5生物活性多肽ENPRAF对线虫寿命的影响
由表5和图9可以看出,当喂食多肽ENPRAF的质量浓度为300mg/L时,实验组中线虫的平均寿命分别延长了约10.2%,同时,其半数死亡时间更是均得到了显著性提高(P<0.05),最长寿命时间相比于空白组也分别延长4天。图9中更是可以直观地看到,相同时间点,实验组中线虫存活率明显高于空白组,线虫寿命得到延长。实验证明,实验采用的多肽质量浓度是合适的。其能够有效延缓线虫衰老,提高存活率,而同时也进一步证明多肽ENPRAF延长寿命的作用并非通过抑制线虫繁殖力来实现,因其具有良好的抗氧化功能和较强的清除自由基能力。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 上海铂辉生物科技有限公司;浙江辉肽生命健康科技有限公司
<120> 一种生物活性多肽ENPRAF及其制备方法和应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Glu Asn Pro Arg Ala Phe
1 5
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaaatcctcg cgcatttg 18
<210> 3
<211> 564
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ala Phe Asp Gly Leu Phe Ile His Ser Leu Leu Gln Asp Leu Thr
1 5 10 15
Pro Thr Leu Val Gly Ser Arg Leu Ser Lys Ile Tyr Gln Pro Phe Asp
20 25 30
Gln Asp Leu Val Leu Ile Phe Arg Lys Asn Arg Lys Asn Tyr Gln Phe
35 40 45
Leu Ile Ser Ala Asn Ala Gln Tyr Pro Arg Met Tyr Leu Thr Glu Gln
50 55 60
Thr Ile Asn Asn Pro Asp Lys Ala Pro Ile Phe Val Met Val Leu Arg
65 70 75 80
Lys Tyr Leu Glu Gly Ser Val Leu Gln Ser Ile Glu Gln Val Gly Leu
85 90 95
Asp Arg Ile Thr Asn Phe His Phe Ser Asn Arg Asn Glu Leu Gly Asp
100 105 110
Glu Val Glu Leu Val Leu Ser Val Glu Val Met Gly Arg His Ser Asn
115 120 125
Val Ile Leu Tyr Asn Gln Lys Asp Asn His Ile Ile Asp Leu Leu Lys
130 135 140
Arg Ile Asn Pro Asp Glu Asn Arg Ala Arg Ile Leu Leu Pro Lys Ala
145 150 155 160
Lys Tyr Glu Leu Pro Pro Leu Lys Pro Gly Leu Asn Gly Leu Thr Leu
165 170 175
Ser Glu Asp Lys Phe Lys Gln Leu Ser Asn Glu Asn Asp Pro Asn Glu
180 185 190
Leu Ser Lys Gln Met Asp Gly Leu Asp Lys Asp Asp Arg Asn Glu Leu
195 200 205
Leu Gly Tyr Leu Glu Asp Asp Tyr Ser Tyr Ser Ser Phe Lys Thr Phe
210 215 220
Phe Asn Gln Phe Glu Asn Pro Arg Ala Phe Val Leu Lys Thr Pro Asn
225 230 235 240
Asn Lys Arg Lys Ile Phe Cys Tyr Leu Pro Tyr His Leu Glu Leu Glu
245 250 255
Lys Glu Ser Ser Asn Pro Asp Leu Asn Lys Gly Leu Asp Glu Phe Tyr
260 265 270
Glu Tyr Gln Ala Asn Arg Asp Trp Val Lys Gln Arg Ala Ser Gln Val
275 280 285
Glu Arg Val Val Lys Asn Glu Gln Lys Lys Leu Ser Lys Lys Ile Lys
290 295 300
Lys Leu Lys Lys Gln Leu Asp Leu Ala Glu Asn Ser Glu Gly Tyr Arg
305 310 315 320
Ile Lys Gly Glu Ile Leu Asn Ala Asn Leu Asn Gln Val Lys Pro Gly
325 330 335
Met Thr Thr Val Ser Leu Pro Asn Tyr Tyr Glu Asn Asn Ala Pro Ile
340 345 350
Glu Ile Lys Leu Asp Pro Ala Leu Ser Pro Thr Arg Asn Ala Gln Lys
355 360 365
Tyr Phe Thr Arg Tyr Lys Lys Leu Arg Asp Ser Ile Lys His Val Asn
370 375 380
Glu Gln Ile Lys Ile Thr Glu Glu Asn Leu Arg Tyr Phe Asp Ser Ile
385 390 395 400
Gln Thr Ala Ile Asp Asn Ala Asp Pro Gln Asp Ile Asp Gln Ile Thr
405 410 415
Asp Glu Leu Ile Asn Gln Gly Tyr Ile Arg Lys Gln Lys Lys Asn Lys
420 425 430
Arg Arg Lys Lys Ile Thr Glu Arg Asn Leu Asn Glu Phe Lys Leu Ser
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Ser Gly Lys His Val Leu Val Gly Lys Asn Asn Tyr Gln Asn Asp Trp
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Leu Thr Leu Lys Lys Ala Asn Lys Ser Asp Tyr Trp Phe His Val Lys
465 470 475 480
Asn Met Pro Gly Ser His Val Ile Leu Arg Asp Asp Gln Pro Ser Asp
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Asp Asp Ile Lys Glu Ala Ala Glu Ile Ala Ala Phe Phe Ser Lys Ala
500 505 510
Lys Asn Ser Thr His Val Gln Val Asp Tyr Val Gln Asp Lys Arg Val
515 520 525
Lys Lys Pro Asn Gly Ala Lys Pro Gly Phe Val Ile Tyr Thr Gly Gln
530 535 540
Asn Ser Ile Glu Val Thr Pro Lys Glu Lys Glu Ile Met Ala Met Lys
545 550 555 560
Val Asn Lys Lys
Claims (9)
1.一种生物活性多肽ENPRAF,其特征在于,其氨基酸序列为Glu-Asn-Pro-Arg-Ala-Phe。
2.编码权利要求1所述生物活性多肽ENPRAF的核苷酸片段,其特征在于,所述核苷酸片段的序列如SEQ ID NO:2所示。
3.如权利要求1所述生物活性多肽ENPRAF的制备方法,其特征在于,通过基因工程的方法人工合成,或直接通过化学合成制备。
4.如权利要求1所述生物活性多肽ENPRAF的应用,其特征在于,所述生物活性多肽ENPRAF在制备具有抗氧化功能的食品、保健品、药物或化妆品中的应用。
5.如权利要求1所述生物活性多肽ENPRAF的应用,其特征在于,所述生物活性多肽ENPRAF在制备具有抗衰老功能的食品、保健品或药物中的应用。
6.如权利要求1所述生物活性多肽ENPRAF的应用,其特征在于,所述生物活性多肽ENPRAF在制备具有抗氧化功能和抗衰老功能的食品、保健品或药物中的应用。
7.一种抗氧化产品,其特征在于,包括如权利要求1所述生物活性多肽ENPRAF;所述的抗氧化产品包括抗氧化食品、抗氧化保健品、抗氧化药物或抗氧化化妆品。
8.一种抗衰老产品,其特征在于,包括如权利要求1所述生物活性多肽ENPRAF;所述的抗衰老产品包括抗衰老食品、抗衰老保健品或抗衰老药物。
9.一种具有抗氧化功能和抗衰老功能的产品,其特征在于,包括如权利要求1所述生物活性多肽ENPRAF;具有抗氧化功能和抗衰老功能的产品包括食品、保健品或药物。
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