CN112481246B - 一种生物活性多肽fspankkltpkky及其制备方法和应用 - Google Patents
一种生物活性多肽fspankkltpkky及其制备方法和应用 Download PDFInfo
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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FSPANKKLTPKKY及其制备方法和应用,生物活性多肽FSPANKKLTPKKY的氨基酸序列为Phe‑Ser‑Pro‑Ala‑Asn‑Lys‑Lys‑Leu‑Thr‑Pro‑Lys‑Lys‑Tyr。经过体外免疫功能调节实验,验证了多肽FSPANKKLTPKKY具有较好的免疫调节功能。本发明的生物活性肽FSPANKKLTPKKY能够显著增强淋巴细胞的体外增殖能力,在一定浓度条件下具有促进巨噬细胞一氧化氮诱生量增加的能力,提高机体抵御外界病原体感染的能力,降低机体发病率,提高生命的质量,对开发具有免疫调节功能的食品、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FSPANKKLTPKKY及其制备方法和应用。
背景技术
近年来,生物活性肽已经成为人们耳熟能详的一个词语。因其具有很多潜在的生物功能,所以引起人们越来越多的关注,成为科学研究的热点之一。目前很多生物活性肽的有益效果也已经得到了很好的证明,如抗癌、降血压、抗菌、降胆固醇、抗糖尿病等等特性。当前最权威的生物活性肽数据库BIOPEP-UMW中已报告了3000多个不同的生物活性肽。
目前对于生物活性肽的研究多集中于食物衍生多肽,对非食物衍生多肽研究和报道较少。且已经有研究结果证实,与食物衍生的生物活性肽相比,非食物衍生的生物活性肽具有更高的亲和力并能有效发挥其生物活性功能。淋巴细胞是免疫系统的中央调节细胞,其大部分功能是由一组被称为淋巴因子的小分子多肽介导的。这些小分子多肽的表达和分泌均是由于抗原刺激的细胞激活而诱导。所以淋巴细胞是动物机体内产生免疫调节肽的主要来源。
免疫调节肽是继阿片肽发现后首次从乳中获得并证明其生理活性的一类生物活性多肽。1981年Jolles等人首次发现,利用胰蛋白酶水解人乳蛋白,可以得到一个氨基酸序列为Val-Glu-Pro-Ile-Pro-Tyr的六肽,体外实验证明该肽能够增强小鼠腹腔巨噬细胞对绵羊红细胞的吞噬作用。Migliore-Samour等人发现来自酪蛋白的六肽Thr-Thr-Met-Pro-Leu-Trp能够刺激绵羊血红细胞对小鼠腹膜巨噬细胞的吞噬作用以及增强对于肺炎克雷伯菌的抵抗,具有抗炎功能。李素萍等人用合成的小鼠骨髓巨噬细胞来与源肽(PGPIPN)饲喂大鼠发现大鼠腹腔巨噬细胞的吞噬作用和红细胞相关的抗炎功能有显著的增强。Bowdis等人在研究来源于牛中性粒细胞的13氨基酸肽indolicidin的免疫功能时发现,多肽indolicidin在巨噬细胞样细胞系中抑制LPS诱导的TNF-α产量。
免疫调节肽一般是指具有免疫调节活性的分子量相对较小的小肽。现在公开的免疫调节肽一般是从蛋白质中酶解分离得到或化学方法合成得到的具有特定免疫调节活性的小肽。但是当这些小肽未从蛋白质中酶解分离时,这些蛋白质本身往往是不具备免疫调节活性的。如何从已知氨基酸序列的种类繁多的蛋白质中寻找具有特定功能的生物活性肽,并研究这些多肽的功能是蛋白领域研究的方向之一。
Protein disulfide-isomerase A3蛋白的氨基酸序列如SEQ ID NO:2所示。目前,现有技术中并没有关于Protein disulfide-isomerase A3蛋白多肽片段相关功能的研究。
发明内容
本发明的目的在于提供一种生物活性多肽FSPANKKLTPKKY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FSPANKKLTPKKY,其氨基酸序列为Phe-Ser-Pro-Ala-Asn-Lys-Lys-Leu-Thr-Pro-Lys-Lys-Tyr,如SEQ ID NO:1所示。
较优的,所述生物活性多肽为小鼠脾脏来源淋巴细胞肽。具体来源于Proteindisulfide-isomerase A3蛋白,并且为Protein disulfide-isomerase A3蛋白第455~467位的氨基酸残基。Protein disulfide-isomerase A3蛋白氨基酸序列如SEQ ID NO:2所示。
Protein disulfide-isomerase A3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Protein disulfide-isomerase A3蛋白第455~467位氨基酸残基的核苷酸片段能编码成熟的生物活性多肽FSPANKKLTPKKY。
较优的,所述生物活性多肽具有抗炎功能和免疫调节功能。
本发明还提供编码所述生物活性肽FSPANKKLTPKKY的多核苷酸。
本发明第二方面,提供了所述生物活性多肽FSPANKKLTPKKY的制备方法,可以通过基因工程的方法人工合成,可以从细胞中通过分离纯化的方法直接获得,可以直接通过化学合成制备。
通过基因工程的方法人工合成所述生物活性肽FSPANKKLTPKKY是本领域技术人员能够实现的技术方案,例如可以是以DNA重组技术为基础,通过合适的DNA模板来控制多肽的序列合成。
关于从细胞中通过分离纯化的方法直接获得的方式可以为:基于给定的生物活性肽FSPANKKLTPKKY的氨基酸序列,采用生物学技术上常规的酶解、纯化方法从小鼠脾脏来源淋巴细胞获得所述生物活性肽FSPANKKLTPKKY。
本发明第三方面,提供了所述生物活性多肽FSPANKKLTPKKY在制备具有抗炎功能的药物或化妆品中的应用。
进一步地,所述生物活性肽FSPANKKLTPKKY在制备促巨噬细胞一氧化氮诱生量的药物中的应用。
本发明第四方面,提供了所述生物活性肽FSPANKKLTPKKY在制备具有免疫调节功能的食物或药物中的应用。
进一步地,所述生物活性肽FSPANKKLTPKKY在制备促进体外淋巴细胞增殖的食物或药物中的应用。
本发明第五方面,提供了一种抗炎产品,包括所述生物活性多肽FSPANKKLTPKKY或所述生物活性多肽FSPANKKLTPKKY的衍生物;所述的抗炎产品包括抗炎药物或抗炎化妆品。
本发明第六方面,提供了一种具有免疫调节功能的产品,包括所述生物活性肽FSPANKKLTPKKY或所述生物活性肽FSPANKKLTPKKY的衍生物;所述具有免疫调节功能的产品包括具有免疫调节功能的食品或具有免疫调节功能的药物。
所述生物活性肽FSPANKKLTPKKY的衍生物是指具有与所述生物活性肽FSPANKKLTPKKY相同的活性或更优的活性。
所述生物活性多肽FSPANKKLTPKKY的衍生物,是指在生物活性多肽FSPANKKLTPKKY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FSPANKKLTPKKY的有益效果为:本发明的生物活性多肽FSPANKKLTPKKY具有较好的抗炎活性;本发明的生物活性肽FSPANKKLTPKKY能够显著增强淋巴细胞的体外增殖能力,在一定浓度条件下具有促进巨噬细胞一氧化氮诱生量增加的能力,提高机体抵御外界病原体感染的能力,降低机体发病率,提高生命的质量,对开发具有免疫调节功能的食品、保健品和药物具有十分重要的意义。
附图说明
图1:质荷比为381.2245的片段的一级质谱图(m/z= 381.2245);
图2:质荷比为381.2245的片段的二级质谱图和多肽az、by断裂情况;
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,1989 and Third edition,2001 ;Ausubel 等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley & Sons,New York,1987 and periodic updates ;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego ;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998 ;METHODSIN ENZYMOLOGY,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FSPANKKLTPKKY的人工合成
一、生物活性肽的合成
人工合成生物活性肽FSPANKKLTPKKY。
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
Time(min) %A %B
0 95.0 5.0
1.50 80.0 20.0
3.50 60.0 40.0
5.00 40.0 60.0
7.00 15.0 85.0
8.00 0.0 100.0
11.00 0.0 100.0
11.50 95.0 5.0
13.00 95.0 5.0
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FSPANKKLTPKKY进行色谱分析和质谱分析。生物活性肽FSPANKKLTPKKY一级质谱图如图1所示,提取峰的二级质谱图和az、by断裂情况如图2所示,可得此峰的多肽质荷比为381.2245,保留时间是7.89min。
3)结果
由图2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381.2245的片段序列为Phe、Ser、Pro、Ala、Asn、Lys、Lys、Leu、Thr、Pro、Lys、Lys、Tyr(FSPANKKLTPKKY),记为SEQ ID NO:1。该片段与Protein disulfide-isomerase A3蛋白第455~467位的残基序列相对应,Protein disulfide-isomerase A3蛋白氨基酸序列如SEQID NO:2所示。
实施例2 生物活性肽的免疫活性实验
一、生物活性多肽FSPANKKLTPKKY的促巨噬细胞一氧化氮诱生量的测定(Griess法)
1. 实验试剂及仪器:
试剂:实验动物 balb/c小鼠(雄性6-8周龄)脾脏淋巴细胞来源生物活性肽FSPANKKLTPKKY;LPS,购自Sigma公司;中性红染色液,碧云天生物技术研究所生产。
仪器设备:LRH-250F生化培养箱 上海恒科技有限公司;GL-22M高速冷冻离心机上海卢湘仪离心机仪器有限公司; Hera cell 150 CO2 培养箱 Heraeus公司;DragonWellscan MK3酶标仪 Labsystems公司。
2. 试验方法:
加入细胞个数为2×106/ml的细胞悬液100μl/孔,贴壁纯化后加入含肽的RPMI1640完全培养液(10% FBS)200μl/孔,炎症组在24h时加入LPS至终浓度10μg/ml,连续培养48h后,收集培养液上清50μl/孔,在培养液上清中依次添加Griess试剂1和Griess试剂2各50μl/孔,室温反应10分钟后,在540nm波长下测定吸光度值(OD540)。
3. 实验结果及分析:
表1 生物活性多肽FSPANKKLTPKKY促巨噬细胞一氧化氮诱生量的测定
注:*,与阴性对照比较,有显著性差异(P <0.05);
**,与阴性对照组比较,有极显著性差异(P <0.01)
实验结果见表1,由表1可知,在实验组中添加生物活性多肽FSPANKKLTPKKY,浓度分别为1 mg/mL和0.5mg/mL,对于正常情况下生长和LPS造炎症情况下生长的促进巨噬细胞的一氧化氮诱生量均有促进作用,与细胞空白组相比,具有极显著性差异(P<0.01)。当生物活性多肽FSPANKKLTPKKY的添加浓度为0.2 mg/mL,在正常情况和LPS造炎症情况下,也能促进巨噬细胞一氧化氮诱生量的增加,并具有显著性差异(P<0.05)。说明生物活性多肽FSPANKKLTPKKY在一定浓度条件下具有促进巨噬细胞一氧化氮诱生量增加的能力。进一步说明生物活性多肽FSPANKKLTPKKY具有抗炎活性。
二、生物活性多肽FSPANKKLTPKKY的体外淋巴细胞增殖能力实验(MTT法)
1. 实验材料与仪器:
试剂与材料:实验动物 balb/c小鼠(雄性6-8周龄,上海交通大学农业与生物学院动物实验中心);实施例1获得的小鼠脾脏淋巴细胞来源生物活性肽FSPANKKLTPKKY;小鼠淋巴细胞提取液(购自索来宝公司);RPMI1640培养基(购自GIBCO公司);3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT,购自Amresco公司);伴刀豆蛋白(ConA,购自Sigma公司);牛血清白蛋白(BSA,购自Genebase公司);胃蛋白酶(购自Sigma公司);胰酶(Corolase PP,购自AB公司)。
仪器设备:LRH-250F生化培养箱,上海恒科技有限公司;GL-22M高速冷冻离心机,上海卢湘仪离心机仪器有限公司;Hera cell 150 CO2 培养箱,Heraeus公司;DragonWellscan MK3酶标仪,Labsystems公司;ALPHA 1-2-LD 真空冷冻干燥机,Christ 公司;超高效液相色谱-四极杆飞行时间质谱仪,waters公司。
2. 实验方法:
无菌条件下取小鼠脾脏,用淋巴细胞提取液提取小鼠淋巴细胞,进行原代培养。用完全RPMI1640培养液将细胞密度调整为2.5×106个/mL。在96孔细胞培养板中依次加入:100μL小鼠淋巴细胞悬液,100μL RPMI1640完全培养液,20μL伴刀豆蛋白,100μL多肽样品。另外,设置空白对照组(pH7.2~7.4,3mol/L的PBS)和阴性对照组(500μg/mL BSA),研究表明其对于体外淋巴细胞增殖没有影响。每组3个平行实验样。在5%CO2 37℃培养箱中培养68h后,无菌条件下每孔加入20μL MTT,继续培养4h,小心弃去上清液,每孔加入100μL二甲基亚砜,37℃生化培养箱孵化10min,摇匀,用酶标仪在570nm处测定吸光值。
体外淋巴细胞增殖能力用刺激指数来表示,计算方法如下:
式中:A1为空白对照在570nm处下的吸光值;A 2为阴性对照组在570nm处下的吸光值,A 3为实验组在570nm处下的吸光值。
3. 实验结果及分析:
表2 生物活性多肽FSPANKKLTPKKY对体外淋巴细胞增殖的影响
实验分组 | 刺激指数SI |
BSA | 1 |
FSPANKKLTPKKY | 1.374±0.028** |
注:**号标记为与阴性对照比较,有极显著性差异(P<0.01)。
实验结果见表2。由表2可知,在生物活性肽FSPANKKLTPKKY的质量浓度为100μg/mL的条件下,生物活性肽FSPANKKLTPKKY的刺激指数大于BSA,说明FSPANKKLTPKKY一定程度上能刺激体外小鼠淋巴细胞的增殖。并且FSPANKKLTPKKY的刺激指数达到了1.374,和阴性对照组具有极显著差异(P<0.01)。因此,可以认定该活性多肽FSPANKKLTPKKY具有显著促进小鼠淋巴细胞增殖的能力,可以作为一种具有免疫调节活性的物质添加到保健品中,能够提高人体的免疫力。
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> 熊猫乳品集团股份有限公司,浙江辉肽生命健康科技有限公司
<120> 一种生物活性多肽FSPANKKLTPKKY及其制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Phe Ser Pro Ala Asn Lys Lys Leu Thr Pro Lys Lys Tyr
1 5 10
<210> 2
<211> 505
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Arg Phe Ser Cys Leu Ala Leu Leu Pro Gly Val Ala Leu Leu Leu
1 5 10 15
Ala Ser Ala Arg Leu Ala Ala Ala Ser Asp Val Leu Glu Leu Thr Asp
20 25 30
Glu Asn Phe Glu Ser Arg Val Ser Asp Thr Gly Ser Ala Gly Leu Met
35 40 45
Leu Val Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Arg Leu Ala
50 55 60
Pro Glu Tyr Glu Ala Ala Ala Thr Arg Leu Lys Gly Ile Val Pro Leu
65 70 75 80
Ala Lys Val Asp Cys Thr Ala Asn Thr Asn Thr Cys Asn Lys Tyr Gly
85 90 95
Val Ser Gly Tyr Pro Thr Leu Lys Ile Phe Arg Asp Gly Glu Glu Ala
100 105 110
Gly Ala Tyr Asp Gly Pro Arg Thr Ala Asp Gly Ile Val Ser His Leu
115 120 125
Lys Lys Gln Ala Gly Pro Ala Ser Val Pro Leu Arg Thr Glu Glu Glu
130 135 140
Phe Lys Lys Phe Ile Ser Asp Lys Asp Ala Ser Val Val Gly Phe Phe
145 150 155 160
Arg Asp Leu Phe Ser Asp Gly His Ser Glu Phe Leu Lys Ala Ala Ser
165 170 175
Asn Leu Arg Asp Asn Tyr Arg Phe Ala His Thr Asn Ile Glu Ser Leu
180 185 190
Val Lys Glu Tyr Asp Asp Asn Gly Glu Gly Ile Thr Ile Phe Arg Pro
195 200 205
Leu His Leu Ala Asn Lys Phe Glu Asp Lys Thr Val Ala Tyr Thr Glu
210 215 220
Lys Lys Met Thr Ser Gly Lys Ile Lys Lys Phe Ile Gln Asp Ser Ile
225 230 235 240
Phe Gly Leu Cys Pro His Met Thr Glu Asp Asn Lys Asp Leu Ile Gln
245 250 255
Gly Lys Asp Leu Leu Thr Ala Tyr Tyr Asp Val Asp Tyr Glu Lys Asn
260 265 270
Ala Lys Gly Ser Asn Tyr Trp Arg Asn Arg Val Met Met Val Ala Lys
275 280 285
Lys Phe Leu Asp Ala Gly His Lys Leu Asn Phe Ala Val Ala Ser Arg
290 295 300
Lys Thr Phe Ser His Glu Leu Ser Asp Phe Gly Leu Glu Ser Thr Thr
305 310 315 320
Gly Glu Val Pro Val Val Ala Ile Arg Thr Ala Lys Gly Glu Lys Phe
325 330 335
Val Met Gln Glu Glu Phe Ser Arg Asp Gly Lys Ala Leu Glu Gln Phe
340 345 350
Leu Gln Glu Tyr Phe Asp Gly Asn Leu Lys Arg Tyr Leu Lys Ser Glu
355 360 365
Pro Ile Pro Glu Ser Asn Glu Gly Pro Val Lys Val Val Val Ala Glu
370 375 380
Asn Phe Asp Asp Ile Val Asn Glu Glu Asp Lys Asp Val Leu Ile Glu
385 390 395 400
Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Asn Leu Glu Pro Lys Tyr
405 410 415
Lys Glu Leu Gly Glu Lys Leu Ser Lys Asp Pro Asn Ile Val Ile Ala
420 425 430
Lys Met Asp Ala Thr Ala Asn Asp Val Pro Ser Pro Tyr Glu Val Lys
435 440 445
Gly Phe Pro Thr Ile Tyr Phe Ser Pro Ala Asn Lys Lys Leu Thr Pro
450 455 460
Lys Lys Tyr Glu Gly Gly Arg Glu Leu Asn Asp Phe Ile Ser Tyr Leu
465 470 475 480
Gln Arg Glu Ala Thr Asn Pro Pro Ile Ile Gln Glu Glu Lys Pro Lys
485 490 495
Lys Lys Lys Lys Ala Gln Glu Asp Leu
500 505
Claims (3)
1.一种生物活性多肽FSPANKKLTPKKY,其特征在于,其氨基酸序列为Phe-Ser-Pro-Ala-Asn-Lys-Lys-Leu-Thr-Pro-Lys-Lys-Tyr。
2.编码权利要求1所述生物活性肽FSPANKKLTPKKY的多核苷酸。
3.如权利要求1所述生物活性多肽FSPANKKLTPKKY的制备方法,其特征在于,直接通过化学合成制备。
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