CN116041485A - 一种具有免疫调节活性的糖肽及应用 - Google Patents
一种具有免疫调节活性的糖肽及应用 Download PDFInfo
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Abstract
本发明公开了一种具有免疫调节活性的糖肽及应用。所述糖肽来源于鸡卵转铁蛋白,包括14种N‑连接结构。所述糖肽的肽段部分包括TAGWVIPMGLIHNR和FGVNGSEK,糖基化位点包括492和637,糖链中含有N‑乙酰葡萄糖胺、甘露糖、半乳糖。所述糖肽肽段TAGWVIPMGLIHNR的糖基化位点为492,N‑连接糖链12种,肽段FGVNGSEK的糖基化位点为637,N‑连接糖链2种。本发明所提供的具有免疫调节的糖肽能够促进小鼠巨噬细胞RAW264.7分泌TNF‑ɑ。转录组测序以及蛋白质组学和磷酸化修饰组学分析显示,糖肽组和空白组细胞的差异表达基因、蛋白、磷酸化蛋白主要集中在经典的免疫信号通路,如“TNF signaling pathway”,“NF‑kappa B signaling pathway”等。本发明的糖肽具有高生物相容性、耐受性以及强大的安全性,可作为一种很有前途的候选佐剂。
Description
技术领域
本发明涉及生物领域中一种具有免疫调节活性的糖肽及应用。
背景技术
蛋白质糖基化是一种非常普遍、复杂和多样的蛋白质翻译后修饰。糖蛋白广泛分布于生命体中,有超过50%的蛋白质都发生了糖基化修饰。根据连接氨基酸残基不同,糖基化主要分为N-糖基化和O-糖基化两大类。在N-糖基化中,糖链通过N-乙酰葡萄糖胺(GlcNAc)与天冬酰胺(Asn)侧链氨基共价结合,其糖链具有五糖核心结构,主要分为复杂型、高甘露糖型、杂合型,糖基化位点具有特定肽段序列Asn-X-Ser/Thr(X≠pro)。基于质谱的蛋白质组学技术为全面分析蛋白质及其修饰提供了有效的手段。针对糖基化修饰研究的技术策略常分为两种,一种是将糖链释放,糖链与修饰位点分别解析;另一种则是直接开展完整糖肽的结构解析。其中,完整N-糖肽更有利于全方位地研究N-糖蛋白的组成和结构。
卵转铁蛋白是鸡蛋清一种具有铁结合和释放能力的天然糖蛋白质,由686个氨基酸组成,分子量大约为76kDa,包含2.6%的糖基成分。糖蛋白具有多糖和蛋白质的共性,能够改善免疫系统、抑制肿瘤、抗氧化、防衰老、降血糖。越来越多的证据表明,糖蛋白在免疫识别中起着关键作用,并且这种特性与糖基化的结构多样性有关。体外(细胞系)和体内(动物模型)测定是用于评估食源性蛋白及其水解物免疫调节作用的常用方法,而在各种细胞系中,小鼠巨噬细胞系RAW264.7已经成为使用最广泛的细胞模型。巨噬细胞在先天免疫和适应性免疫中发挥重要作用,是形成抵御体内外部或内部危险信号的第一导防线,具有吞噬细胞、清除机体损伤处组织和细胞碎片以及病原体等功能。蛋白质在细胞分子机制中占很大一部分,它执行各种细胞活动,决定细胞命运。蛋白质组学能够在大规模水平上研究不同条件下蛋白质特征,并由此获得蛋白质水平上关于疾病机理、细胞代谢等过程的整体而全面的认识。近年来,基于质谱的蛋白质组学被广泛应用于了解分子和信号通路。此外,越来越多的研究发现,许多重要的生命活动、疾病发生不仅与蛋白质的丰度相关,更重要的是被各类蛋白质翻译后修饰(PTM)所调控。蛋白质的翻译后修饰,如磷酸化,在宿主的先天免疫反应中起着至关重要的作用。
传统的糖肽鉴定程序都有部分局限性,很难从一个给定的位点识别出所有的多糖,也很难量化它们的相对丰度。目前糖肽在免疫调控方面的机理研究不够广泛、深入,基于蛋白质组学和磷酸化修饰组学能够找出哪些蛋白、磷酸化蛋白发生了变化,以及这些变化对巨噬细胞的免疫调控产生了哪些影响。
发明内容
本发明公开的一种具有免疫调节活性的糖肽,其获得包括,利用胃蛋白酶Pepsin、胰蛋白酶Trysin和糜蛋白酶α-Chymotrypsin对蛋清中的卵转铁蛋白进行酶解,Con ASepharoseTM 4B富集完整N-糖肽,C18-RPLC-MS/MS(HCD)鉴定分析,最后根据糖肽质谱信息进行鉴定。
本发明所提供的具有免疫调节的糖肽,有14种N-连接结构。所述糖肽的肽段部分包括TAGWVIPMGLIHNR和FGVNGSEK,糖基化位点包括492和637,糖链中含有N-乙酰葡萄糖胺、甘露糖、半乳糖。所述糖肽肽段TAGWVIPMGLIHNR的糖基化位点为492,N-连接糖链12种,肽段FGVNGSEK的糖基化位点为637,N-连接糖链2种。
本发明所提供的具有免疫调节的糖肽,当糖肽的浓度为125ug/ml时,能够促进小鼠巨噬细胞RAW264.7分泌TNF-ɑ。转录组测序以及蛋白质组学和磷酸化修饰组学分析显示,糖肽组和空白组细胞的差异表达基因、蛋白、磷酸化蛋白主要集中在经典的免疫信号通路,如“TNF signaling pathway”,“NF-kappa B signaling pathway”等。
本发明的目的通过以下技术方案实现:
(1)糖肽的富集与鉴定
取鸡卵转铁蛋白溶于PBS(3X)(1:2w/w),向所得混合液加入2倍等体积的去离子水,用1M HCL调节PH至2.0,加胃蛋白酶Pepsin(1:25w/w),在37℃恒温水浴2h,每30min搅拌一次。用1M NaOH将水解产物调节PH至7.8,加入胰蛋白酶Trysin(1:25w/w)和糜蛋白酶α-Chymotrypsin(1:100w/w),在37℃恒温水浴6h,每30min搅拌一次。所得的消化液用0.25um的微孔滤膜过滤,收集滤过液。
Con A SepharoseTM 4B亲和层析柱在pH 6.0含有1M NaCl,1mM CaCl2,1mM MnCl2和1mM MgCl2的0.1M乙酸盐缓冲液中预溶胀30min,用10倍柱体积pH7.4的20mM Tris-HCl结合缓冲液冲洗填料。滤过液以2倍柱体积进样后,先用结合缓冲液10倍柱体积冲洗,然后依次用含0.2M与0.5M的α-D-甲基甘露糖苷的结合缓冲液洗脱,收集4倍柱体积的0.2M洗脱液。洗脱结束后用5倍柱体积的结合缓冲液冲洗,并将填料保存于含20%乙醇的结合缓冲液中。
将0.2M的洗脱液放入500-1000D的透析袋中进行透析,控制温度4℃,时间36h,样品与透析液1:200,换液3次。洗脱液透析后,真空浓缩至干。
基于C18-RPLC-MS/MS(HCD)鉴定分析,以卵转铁蛋白序列和理论质谱碎片信息、N-糖链核心骨架结构及其理论质谱碎片信息为参照,将实验获得的糖肽一级质谱荷质比、二级质谱碎片信息与之匹配,推定糖肽完整结构。
(2)免疫调节活性的测定
建立巨噬细胞模型,检测糖肽对巨噬细胞RAW264.7的毒性影响以及对细胞因子TNF-ɑ产生的影响,并基于转录组测序以及蛋白质组学和磷酸化修饰组学,分析糖肽对RAW264.7的免疫调节活性。
用不同浓度的糖肽刺激RAW264.7细胞8h,对照组细胞仅在培养基中培养,细胞处理完成后,检测其活力,选择最佳的糖肽作用浓度,并检测糖肽作用RAW264.7细胞后上清液中TNF-α的含量。RAW264.7细胞诱导8h后,收集细胞沉淀并从中提取total RNA,利用Nanodrop 2000对所提RNA的浓度和纯度进行检测,最后再基于Illumina Novaseq 6000平台测序。RAW264.7细胞诱导8h后,收集细胞沉淀并从中提取蛋白,利用胰酶将蛋白酶解,肽段经TMT标记和HPLC分级,(磷酸化修饰组再经IMAC材料富集后)使用EASY-nLC 1200超高效液相系统进行分离,肽段分离后被注入NSI离子源中进行电离然后进Q ExactiveTMHF-X质谱进行分析。
本发明与现有技术相比,具有如下有益效果:
本发明从鸡卵转铁蛋白中富集到了一种具有免疫调节活性的糖肽,所述糖肽来源于鸡卵转铁蛋白,包括14种N-连接结构。所述糖肽的肽段部分包括TAGWVIPMGLIHNR和FGVNGSEK,糖基化位点包括492和637,糖链中含有N-乙酰葡萄糖胺、甘露糖、半乳糖。所述糖肽肽段TAGWVIPMGLIHNR的糖基化位点为492,N-连接糖链12种,肽段FGVNGSEK的糖基化位点为637,N-连接糖链2种。糖肽能够促进小鼠巨噬细胞RAW264.7分泌TNF-ɑ,糖肽组和空白组细胞的差异表达基因、蛋白、磷酸化蛋白主要集中在经典的免疫信号通路,如“TNFsignaling pathway”,“NF-kappa B signaling pathway”等。本发明的糖肽具有高生物相容性、耐受性以及强大的安全性,可作为一种很有前途的候选佐剂。
附图说明
图1肽段TAGWVIPMGLIHNR的ASN492位点上的一种图形解离图。
图2肽段FGVNGSEK的ASN637位点上的一种图形解离图。
图3糖肽对RAW264.7巨噬细胞TNF-α分泌量的影响。
具体实施方式
下面的实施例对本发明作详细的说明。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中未注明具体条件的实验方法,通常按照常规条件,或按照厂家建议的条件。
实施例1.糖肽的富集与鉴定
取鸡卵转铁蛋白溶于PBS(3X)(1:2w/w),向所得混合液加入2倍等体积的去离子水,用1M HCL调节PH至2.0,加胃蛋白酶Pepsin(1:25w/w),在37℃恒温水浴2h,每30min搅拌一次。用1M NaOH将水解产物调节PH至7.8,加入胰蛋白酶Trysin(1:25w/w)和糜蛋白酶α-Chymotrypsin(1:100w/w),在37℃恒温水浴6h,每30min搅拌一次。所得的消化液用0.25um的微孔滤膜过滤,收集滤过液。
Con A SepharoseTM 4B亲和层析柱在PH 6.0含有1M NaCl,1mM CaCl2,1mM MnCl2和1mM MgCl2的0.1M乙酸盐缓冲液中预溶胀30min,用10倍柱体积PH7.4的20mM Tris-HCl结合缓冲液冲洗填料。滤过液以2倍柱体积进样后,先用结合缓冲液10倍柱体积冲洗,然后依次用含0.2M与0.5M的α-D-甲基甘露糖苷的结合缓冲液洗脱,收集4倍柱体积的0.2M洗脱液。洗脱结束后用5倍柱体积的结合缓冲液冲洗,并将填料保存于含20%乙醇的结合缓冲液中。
将0.2M的洗脱液放入500-1000D的透析袋中进行透析,控制温度4℃,时间36h,样品与透析液1:200,换液3次。洗脱液透析后,真空浓缩至干。
基于C18-RPLC-MS/MS(HCD)鉴定分析,以卵转铁蛋白序列和理论质谱碎片信息、N-糖链核心骨架结构及其理论质谱碎片信息为参照,将实验获得的糖肽一级质谱荷质比、二级质谱碎片信息与之匹配,推定糖肽完整结构。
结果显示,在卵转铁蛋白中共鉴定出14个完整N-糖肽结构,这些糖肽结构涉及2个N-糖基化位点。表1是卵转铁蛋白完整N-糖肽的部分糖基化信息。TAGWVIPMGLIHNR的Asn-492位点上有12种N-糖链结构,FGVNGSEK的Asn-637位点上有2种N-糖链结构。在这14种N-糖链结构中,包含1种核心结构,3种高甘露糖型,8种复杂型和2种混合型。如图1和图2所示,糖肽TAGWVIPMGLIHNR离子强度最高的是多糖组成为N2H5的一个高甘露糖三天线糖链结构,糖肽FGVNGSEK离子强度最高是多糖组成为N6H3的一个复杂四天线糖链结构。
表1:卵转铁蛋白完整N-糖肽的部分糖基化信息
实施例2.免疫调节活性的测定
建立巨噬细胞模型,检测糖肽对巨噬细胞RAW264.7的毒性影响以及对细胞因子TNF-ɑ产生的影响,并基于转录组测序以及蛋白质组学和磷酸化修饰组学,分析糖肽对RAW264.7的免疫调节活性。
用不同浓度的糖肽刺激RAW264.7细胞8h,对照组细胞仅在培养基中培养,细胞处理完成后,检测其活力,选择最佳的糖肽作用浓度,并检测糖肽作用RAW264.7细胞后上清液中TNF-α的含量。RAW264.7细胞诱导8h后,收集细胞沉淀并从中提取total RNA,利用Nanodrop 2000对所提RNA的浓度和纯度进行检测,最后再基于Illumina Novaseq 6000平台测序。RAW264.7细胞诱导8h后,收集细胞沉淀并从中提取蛋白,利用胰酶将蛋白酶解,肽段经TMT标记和HPLC分级,(磷酸化修饰组再经IMAC材料富集后)使用EASY-nLC 1200超高效液相系统进行分离,肽段分离后被注入NSI离子源中进行电离然后进Q ExactiveTMHF-X质谱进行分析。
细胞毒性实验显示,125μg/mL的糖肽对RAW264.7细胞活力影响较小,更高浓度的糖肽表现出一定的抑制作用,因此,选择125μg/mL作为后续细胞实验的处理浓度。图3表明,糖肽(125μg/mL)处理显著增强了RAW264.7的TNF-α分泌(1254.81%),并且呈现剂量依赖性(p<0.0001)。转录组测序显示:与空白组相比,鉴定出糖肽组中差异表达基因共1715个,上调的差异表达基因有1112个,下调的差异表达基因有603个。差异表达基因的KEGG富集分析结果表明,糖肽处理激活了“TNF signaling pathway”、“NF-kappa B signalingpathway”、“MAPK signaling pathway”等关键信号通路。蛋白质组学显示:糖肽组和空白组细胞,差异蛋白共有184个,其中149个上调,35个下调。这些差异蛋白,富集到76条KEGG通路,上调的差异蛋白富集途径包括“Cytokine-cytokine receptor interaction”、“NF-kappa B signaling pathway”、“TNF signaling pathway”等,下调的差异蛋白富集在“Arrhythmogenic right ventricular cardiomyopathy”、“ECM-receptor interaction”等KEGG通路中。磷酸化修饰组学显示:糖肽组和空白组细胞,差异磷酸化蛋白共有625个,其中242个上调,383个下调,另有774个差异表达磷酸化修饰位点被鉴定到,495个上调,279个下调。差异磷酸化蛋白被富集到8条KEGG通路,上调的差异磷酸化蛋白富集的途径包括“Mitophagy-animal”、“Focal adhesion”、“Leukocyte transendothelial migration”,下调的差异磷酸化蛋白富集的途径包括“Choline metabolism in cancer”、“Fc gamma R-mediated phagocytosis”、“Lysine degradation”等。
实施例3.免疫调节活性糖肽的应用
在实际生产中,糖肽可以作为佐剂添加在疫苗中,诱发机体产生长期、高效的特异性免疫反应,提高机体保护能力,减少免疫物质的用量,降低疫苗的生产成本。
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,这种描述没有限制性,实际的实施方式并不局限于此。总而言之,如果本领域的普通技术人员受其启示,在不脱离本发明创造宗旨的情况下,不经创造性的设计出与该技术方案相似的结构方式及实施例,均应属于本发明的保护范围。
Claims (7)
1.一种具有免疫调节活性的糖肽,其特征在于,所述糖肽来源于鸡卵转铁蛋白,所述糖肽具有序列表1-2所示的2个肽段,包括TAGWVIPMGLIHNR和FGVNGSEK,所述糖肽具有14种N-连接结构,糖基化位点包括492和637,糖链中含有N-乙酰葡萄糖胺、甘露糖、半乳糖。
2.根据权利要求1所述的一种具有免疫调节活性的糖肽,其特征在于,所述糖肽肽段1的糖基化位点为492,N-连接糖链12种,肽段2的糖基化位点为637,N-连接糖链2种。
3.权利要求1所述的糖肽在疫苗中的应用。
4.权利要求1-3任意一项所述具有免疫调节活性的糖肽的制备方法,其特征在于:所述制备方法包括以下步骤:
S1:取鸡卵转铁蛋白溶于PBS,向所得混合液加入2倍等体积的去离子水,用1M HCL调节pH至2.0,加胃蛋白酶Pepsin,在37℃恒温水浴2h,每30min搅拌一次;
S2:用1M NaOH将水解产物调节PH至7.8,加入胰蛋白酶Trysin和糜蛋白酶α-Chymotrypsin,在37℃恒温水浴6h,每30min搅拌一次;
S3:所得的消化液用0.25um的微孔滤膜过滤,收集滤过液;
S4:滤过液经Con A SepharoseTM4B亲和层析柱富集完整N-糖肽并梯度洗脱;
S5:洗脱液透析后,真空浓缩至干。
5. 根据权利要求4所述具有免疫调节活性的糖肽的制备方法,其特征在于:所述步骤S1:卵转铁蛋白与PBS 的质量比为1:2,调节pH 2.0后加入胃蛋白酶Pepsin;所述步骤S2:调节pH 7.8后加入胰蛋白酶Trysin和糜蛋白酶α-Chymotrypsin。
6. 根据权利要求4所述具有免疫调节活性的糖肽的制备方法,其特征在于:所述步骤S4:Con A SepharoseTM 4B亲和层析柱在pH 6.0含有1M NaCl,1mM CaCl2,1mM MnCl2和1mMMgCl2的0.1M乙酸盐缓冲液中预溶胀30min,用10倍柱体积pH7.4的20 mM Tris-HCl结合缓冲液冲洗填料;
S3中滤过液以2倍柱体积进样后,先用结合缓冲液10倍柱体积冲洗,然后依次用含0.2M与0.5M的α-D-甲基甘露糖苷的结合缓冲液洗脱,收集4倍柱体积的0.2 M洗脱液,洗脱结束后用5倍柱体积的结合缓冲液冲洗,并将填料保存于含20%乙醇的结合缓冲液中。
7.根据权利要求4所述具有免疫调节活性的糖肽的制备方法,其特征在于:所述步骤S5:将0.2M的洗脱液放入500-1000D的透析袋中进行透析,控制温度4℃,时间36h,样品与透析液1:200,换液3次。
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