CN116987200B - 一种识别外泌体的融合蛋白及其应用 - Google Patents
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Abstract
本发明属于外泌体富集技术领域,公开了一种识别外泌体的融合蛋白及其应用。本发明提供一种识别外泌体的融合蛋白,包括支架蛋白和外泌体结合肽;所述支架蛋白包括hBD3蛋白和/或Syncytin‑1蛋白N端;所述hBD3蛋白和所述Syncytin‑1蛋白N端由linker序列连接;在所述支架蛋白的N末端、内部或C末端融合表达所述外泌体结合肽。本发明的融合蛋白能够识别外泌体膜蛋白多个膜蛋白,且无需像多肽人工合成,或纯化抗体等高成本复杂操作。本发明通过大量表达制备能够识别外泌体的融合蛋白,并将其固定于磁性粒子表面,利用其识别外泌体及磁性分离特性,便捷经济收集外泌体,进行后续研究或应用。
Description
技术领域
本发明涉及外泌体富集技术领域,具体涉及一种识别外泌体的融合蛋白及其应用。
背景技术
外泌体为细胞分泌到胞外的纳米囊泡,其粒径约为30nm~160nm。外泌体具有双层膜结构,电镜下呈现茶托状形态。外泌体含有蛋白、核酸及脂质等,可被细胞摄取进入胞内,参与细胞间的分子传递及通讯。外泌体广泛参与了细胞物质信息运输,调控细胞生理功能,能够进行抗原呈递、促进肿瘤发生转移等。不同细胞分泌的外泌体在数量和内含物差异性大,外泌体膜蛋白CD9、CD63、CD81及内部蛋白TSG101、HSP90作为其通用标记,后续发现膜蛋白CD47、SLC1A5、SLC3A2、ITGB1等也在外泌体普遍存在。不同来源外泌体特异性内含物可以作为其来源细胞的标记,作为疾病早期诊断标记物,目前已有报道通过外泌体分析获得对相应肿瘤的预测。此外,通过工程化改造外泌体,或电转化、化学点击等方法搭载药物分子,用于疾病的治疗。
外泌体分离富集是外泌体用于诊断及应用的重要前提。超速离心是外泌体分离的金标准。随着研究深入,多种方法被应用于外泌体分离富集,包括切向流离心、密度梯度离心、排阻柱层析、微流控芯捕获等方法。然而,这些方法需要价格昂贵精密仪器,在仪器购买、空间和操作层面有诸多限制,且富集效果差。现有技术公开了利用蛋白或肽识别外泌体并进行富集的方法。利用外泌体膜蛋白抗体,结合Protein A/G beads,或固定外泌体膜蛋白抗体beads,进行免疫亲和富集外泌体。然而,该方法中抗体生产成本高,是限制该方法普及的重要因素。此外,还可以利用外泌体质膜识别肽,或外泌体膜蛋白特异性结合肽(如特异性识别外泌体膜蛋白CD63蛋白的肽段CP05)固定化beads,亲和捕获外泌体。然而,该方法中肽段依赖体外合成,其所需技术高且成本高,限制该方法的广泛应用。
发明内容
本发明的目的在于克服现有技术的不足之处而提供一种识别外泌体的融合蛋白及其应用。
为实现上述目的,本发明采取的技术方案如下:
第一方面,本发明提供一种识别外泌体的融合蛋白,包括支架蛋白和外泌体结合肽;所述支架蛋白包括hBD3蛋白和/或Syncytin-1蛋白N端;
所述hBD3蛋白和所述Syncytin-1蛋白N端由linker序列连接;
在所述支架蛋白的N末端、内部或C末端融合表达所述外泌体结合肽。
本发明的融合蛋白能够识别外泌体膜蛋白多个膜蛋白,相比脂质结合肽、膜蛋白抗体、膜蛋白结合肽等单个成分或蛋白识别具有优势。所述融合蛋白可单独使用hBD3、Syncytin-1蛋白N端作为支架蛋白,在支架蛋白N末端、C末端或内部融合表达其他外泌体结合肽,所述外泌体结合肽与hBD3、Syncytin-1蛋白N端可以不同数量及不同排序来组合。
作为本发明所述的识别外泌体的融合蛋白的优选实施方式,所述外泌体结合肽包括ITGB1结合肽RGD4C、CD63结合肽CP05。
作为本发明所述的识别外泌体的融合蛋白的优选实施方式,所述linker序列选自3GS linker、G4S linker、EA3K linker中的至少一种。
作为本发明所述的识别外泌体的融合蛋白的优选实施方式,所述融合蛋白的氨基酸序列如SEQ ID NO:1所示。
作为本发明所述的识别外泌体的融合蛋白的优选实施方式,所述融合蛋白还包括蛋白分离标签;所述蛋白分离标签选自His、Strep Tag II、GST、MBP中的至少一种。
第二方面,本发明提供一种编码识别外泌体的融合蛋白的核苷酸,所述核苷酸的碱基序列如SEQ ID NO:2所示。
第三方面,本发明提供一种重组菌,所述重组菌表达上述的融合蛋白。
作为本发明所述的重组菌的优选实施方式,所述重组菌为大肠杆菌。
第四方面,本发明提供一种细胞外泌体的富集方法,包括以下步骤:
(1)诱导所述重组菌表达所述融合蛋白,分离所述融合蛋白;
(2)纯化并变性所述融合蛋白,得变性蛋白;
(3)加入磁珠固化所述变性蛋白;复性所述变性蛋白,得复性磁性粒子;
(4)收集细胞外泌体;将所述复性磁性粒子与所述细胞外泌体共孵育,通过磁性分离富集外泌体。
作为本发明所述的富集方法的优选实施方式,在所述步骤(2)中,所述变性采用7M~9 M尿素或5 M~7 M盐酸胍处理;在所述步骤(3)中,所述复性采用大量稀释复性法、透析、超滤溶液置换法中的至少一种;在所述步骤(4)中,所述收集细胞外泌体采用超速离心、切向流离心、SEC、超滤、密度梯度离心中的至少一种;所述细胞外泌体来自组织体液、原代细胞培养液、细胞系培养液中的至少一种。
与现有技术相比,本发明的有益效果为:
本发明设计了识别外泌体普遍表达膜蛋白的融合蛋白,该融合蛋白能够识别外泌体膜蛋白多个膜蛋白,相比脂质结合肽、膜蛋白抗体、膜蛋白结合肽等单个成分或蛋白识别具有优势。该融合蛋白使用大肠杆菌表达及标签纯化,无需像多肽人工合成,或纯化抗体等高成本复杂操作。在变性条件下该融合蛋白能与链霉亲和素磁珠能够高效组装,抵抗不同溶液条件能力强。该融合蛋白磁珠可通过多次点加至大体系复性溶液中,完成蛋白复性,且复性后蛋白能够在磁珠上稳定保持不脱落并保持活性,与透析、超滤置换溶液等措施便捷且复性效率高。本发明通过大量表达制备能够识别外泌体的融合蛋白,并将其固定于磁性粒子表面,利用其识别外泌体及磁性分离特性,便捷经济收集外泌体,进行后续研究或应用。
附图说明
图1为外泌体捕获磁珠ExoBPmagbeads构建及外泌体磁分离示意图;
图2为pET28a-His-Strep Tag II-ExoBP表达质粒示意图;
图3为His-Strep Tag II-ExoBP融合蛋白示意图;
图4为His-Strep Tag II-ExoBP诱导表达检测;
图5为His-Strep Tag II-ExoBPWB验证;
图6为变性条件下Ni琼脂糖纯化的His-Strep Tag II-ExoBP检测;
图7为变性条件下链霉亲和素磁珠(Samagbeads)结合His-Strep Tag II-ExoBP后形成ExoBPmagbeads的检测;
图8为ExoBPmagbeads通过稀释(dil)和透析(dia)复性后的检测;
图9为脐带间充质干细胞无血清培养液超速离心后SEC HPLC检测;
图10为脐带间充质干细胞外泌体通过ExoBPmagbeads分离后外泌体marker检测;
图11为稀释复性(dil)和透析复性(dia)的ExoBPmagbeads分离脐带间充质干细胞来源外泌体后其marker检测。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例中所用的试验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例:
(1)构建融合蛋白,包括支架蛋白及肽段氨基酸序列。融合蛋白包括人hBD3蛋白(与SLC3A2蛋白结合)、SLC1A5结合蛋白Syncytin-1N端(21-150 aa,与SLC1A5结合)、RGD4C肽(与ITGB1结合)、CP05肽(与CD63结合)、linker序列。
具体的,使用SLC3A2结合蛋白hBD3、SLC1A5结合蛋白Syncytin-1N端(21-150 aa,简写为SN)及3(GS)linker构建融合蛋白主要部分hBD3-3(GS)-SN(支架蛋白),可用于表达外泌体膜蛋白结合肽的支架,可用于融合表达外泌体膜蛋白结合肽段。
分别在上述支架蛋白的N端与C端融合表达ITGB1结合肽RGD4C及CD63结合肽CP05,同时分别加入G4S linker与EA3K linker序列,构建His-Strep Tag II-RGD4C-G4S-hBD3-3(GS)-SN-EA3K-CP05融合蛋白,如图3所示。其中,His可用于Ni柱纯化,Strep Tag II可固定于Samagbeads(链霉亲和素磁珠),hBD3-SN是结合外泌体膜蛋白SLC3A2和SLC1A5的蛋白,RGD4C和CP05能够结合外泌体膜蛋白ITGB1和CD63的肽段。
目标融合蛋白RGD4C-G4S-hBD3-3(GS)-SN-EA3K-CP05的氨基酸序列(SEQ ID NO:1)为:
WSHPQFEKGALEVLFQGPACDCRGDCFCGGGGGSGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKKGSGSGSAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLSKGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQTGMSDGGGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDEAAAKCRHSQMTVTSRL。
根据大肠杆菌密码子偏好性优化后基因序列(SEQ ID NO:2)为:
TGGTCTCATCCGCAATTCGAGAAAGGTGCACTGGAAGTTCTGTTTCAAGGTCCGGCATGTGATTGCCGTGGCGATTGCTTCTGCGGTGGCGGCGGTGGTAGCGGTATTATCAACACTCTGCAGAAATACTACTGCCGTGTACGCGGCGGTCGTTGTGCAGTGCTGAGCTGCCTGCCGAAAGAAGAACAAATCGGCAAATGTTCTACCCGCGGTCGCAAGTGTTGTCGTCGTAAGAAGGGTTCCGGCAGCGGCTCCGCTCCGCCACCGTGCCGCTGCATGACCTCTAGCAGCCCGTATCAAGAATTTCTGTGGCGTATGCAACGTCCGGGCAACATTGATGCACCATCTTACCGTAGCCTGAGCAAAGGTACTCCGACCTTCACCGCTCACACCCACATGCCGCGTAACTGCTATCACTCTGCCACCCTGTGTATGCACGCTAACACTCACTACTGGACTGGTAAAATGATTAACCCGTCTTGCCCGGGCGGTCTGGGCGTTACTGTATGTTGGACTTATTTCACTCAGACTGGCATGAGCGATGGTGGTGGTGTGCAAGACCAGGCTCGTGAGAAACACGTAAAGGAGGTGATCAGCCAGCTGACCCGTGTTCACGGTACTTCCAGCCCGTACAAGGGCCTGGACGAGGCGGCTGCAAAGTGTCGTCACTCCCAGATGACCGTAACTTCTCGCCTG。
具体序列如表1所示:
表1蛋白序列和优化密码子后的基因序列
(2)按照上述序列合成基因并利用PCR扩增得到该基因片段,同时PCR将pET-His-Strep Tag II质粒线性化,将基因片段和线性化质粒利用T4 DNA ligase连接,转化E.coliDH5α感受态,涂布琼脂糖平板,挑选克隆进行PCR验证后测序。将测序正确菌落扩大培养,提取质粒,转化E. coliBL21(DE3)表达系统,挑选测序正确克隆,得到表达菌株pET-His-Strep Tag II-ExoBPE. coliBL21(DE3)。
(3)将上述表达菌株接种于5 mL LB培养基,37℃、200 rpm过夜培养,按照1:100将其转接于100 mL LB培养基,37℃、200 rpm培养约2 h,待其OD 600达到0.5~0.8时,加入终浓度为0.5 mM IPTG,继续培养3 h,离心收集菌体,加入10 mL 8 M Urea PBS,充分振荡室温裂解1.5 h,常温8000 rpm离心15 min收集上清液,此上清液中含有变性的His-StrepTag II-ExoBP蛋白。His-Strep Tag II-ExoBP诱导表达检测如图4所示,加入IPTG诱导后全细胞裂解液(WCL)相比诱导前(-)融合蛋白大量表达(约30kD处),该蛋白在破碎液上清(S)含量少,大部分存在于包涵体中(P)。His-Strep Tag II-ExoBPWB验证如图5所示,利用His标签抗体,可在表达菌株加入IPTG诱导后全细胞裂解液(WCL)中检测到目的蛋白。
(4)向上述液体中加入1 mL Ni琼脂糖,室温混匀1 h,自然沉淀或低速离心弃去未结合液,保留Ni琼脂糖柱体,向其中加入10 mL 8 M Urea PBS洗涤柱体,弃去洗涤液保留柱体,重复一次。加入1 mL 300 mM imidazole/8 M Urea PBS洗脱液充分混匀,室温孵育3min,取出洗脱液,重复3次,洗脱液即为纯化的变性His-Strep Tag II-ExoBP蛋白。变性条件下Ni琼脂糖纯化的His-Strep Tag II-ExoBP检测如图6所示,4次洗脱纯化的蛋白(elution),经SDS-PAGE及考马斯亮蓝染色后,可见单一目的蛋白条带。
(5)取1 mL上述液体,加入20 μL SA magbeads,室温孵育1 h,置于磁力架1 min,弃去液体。向其中加入1 mL 8 M Urea PBS洗涤3 min,置于磁力架1 min,弃去液体,重复洗涤3次。加入20 μL 8 M Urea PBS重悬磁珠,即为变性ExoBPmagbeads。变性条件下链霉亲和素磁珠(Samagbeads)结合His-Strep Tag II-ExoBP后形成ExoBPmagbeads的检测如图7所示,ExoBPmagbeads经SDS-PAGE及考马斯亮蓝染色,可看到ExoBP蛋白条带(约30kD),而BSA与Samagbeads共孵育磁性富集后,相同条件检测磁珠上蛋白,未见BSA蛋白条带,表明ExoBP与Samagbeads特异性结合。其中,Samagbeads上Sa蛋白可在SDS-PAGE被检测到(约15kD)。
(6)取上述ExoBPmagbeads少量多次点加至10 mL PBS中,室温孵育30 min,置于磁力架分离磁珠,弃去液体,加入20 μL PBS重悬磁珠,即通过大体积稀释得复性的His-StrepTag II-ExoBP磁粒子悬液。或将上述ExoBPmagbeads加入1mL8 M Urea PBS重悬,置于透析袋中,将透析袋于500mL4M、2 M、1M、0.5 M、0MUrea PBS中,室温分别透析1h,收集磁珠悬液于磁力架分离,弃去上清,加入PBS洗涤两次,加入20 μL PBS重悬磁珠,即得到透析复性ExoBPmagbeads。ExoBPmagbeads通过复性后的检测如图8所示,ExoBPmagbeads复性前(-)、稀释复性(dil)、透析复性(dia)后SDS-PAGE及考马斯染色显示,复性后磁珠ExoBP含量与复性前无显著差异。
变性条件下利用链霉亲和素磁珠固化含Strep Tag II标签蛋白,将其多次点加入无变性剂溶液中,通过大量稀释复性方法复性蛋白,得到固定于磁珠的复性蛋白,无需透析复性等复杂操作即可得到固定于磁珠的复性蛋白,后续可通过biotin洗脱获得活性蛋白。本实施例中在尿素变性条件下,将His-Strep Tag II-ExoBP蛋白固定于链霉亲和素磁珠,后在大体积PBS稀释该磁珠,得到复性重新折叠的His-Strep Tag II-ExoBP磁珠ExoBPmagbeads。
(7)37℃、5.0% CO2培养人脐带间充质干细胞,当其汇合度至80%~90%左右时,弃去完全培养基,加入无血清培养基继续培养24 h,收集培养基;4℃、200 g离心10 min,取上清4℃、2000 g离心20 min,取上清4℃、10000 g离心40 min,将上清于0.22 μm滤膜过滤,4℃、120000 g离心60 min,弃去上清加入等体积预冷PBS重悬,4℃、120000 g离心60 min,弃去上清,加入预冷200 μL PBS(约30 mL~40 mL培养基收集液外泌体)重悬外泌体,-80℃保存或进行后续操作。
(8)取上述外泌体,利用200ÅSEC柱HPLC进行检测,流动相为PBS,流速设置为0.4mL/min,检测时间20min。利用SEC柱HPLC根据峰图检测外泌体纯化效果如图9所示,外泌体在约第5.5min出峰,在约7min结束。
(9)取20 μL外泌体,加入5 μL ExoBP magbeads,加入PBS至50 μL,4℃充分混匀孵育1 h,置于磁力架分离弃去上清,加入100 μL PBS洗涤,置于磁力架分离去除上清,重复一次,加入20 μL PBS。利用CD63 CD81 TSG101 HSP90-b抗体进行western blotting检测外泌体表面标记。脐带间充质干细胞外泌体通过稀释复性ExoBPmagbeads分离后外泌体marker检测如图10所示,HisTag抗体检测条带为ExoBP蛋白,ExoBPmagbeads吸附富集样品,可检测到外泌体表面蛋白markerCD63 CD81 TSG101 HSP90-b,表明ExoBPmagbeads能够吸附外泌体。利用上述条件,通过稀释复性(dil)和透析复性(dia)的ExoBPmagbeads分离脐带间充质干细胞来源外泌体,westernblotting检测其marker如图11所示,两种复性magbeads吸附外泌体后,均能检测到外泌体markerCD81及HSP90-b,表明两种复性方式ExoBPmagbeads均具有捕获外泌体活性。
本发明设计了识别外泌体普遍表达膜蛋白的融合蛋白,该融合蛋白能够识别外泌体膜蛋白多个膜蛋白,相比脂质结合肽、膜蛋白抗体、膜蛋白结合肽等单个成分或蛋白识别具有优势。该融合蛋白使用大肠杆菌表达及标签纯化,无需像多肽人工合成,或纯化抗体等高成本复杂操作。在变性条件下(8 M尿素),该融合蛋白能与链霉亲和素磁珠能够高效组装,抵抗不同溶液条件能力强。该融合蛋白磁珠可通过多次点加至大体系复性溶液中,完成蛋白复性,且复性后蛋白能够在磁珠上稳定保持不脱落并保持活性,与透析、超滤置换溶液等措施便捷且复性效率高。此外,本发明的融合蛋白可单独使用hBD3、Syncytin-1N端作为支架蛋白,融合表达其他外泌体结合肽,结合肽与hBD3、Syncytin-1可以不同数量及不同排序来组合;除了使用大肠杆菌表达系统pET28a+质粒及E.coliBL21(DE3),也可使用其他大肠杆菌表达系统,或真核表达系统;融合蛋白表达纯化His、Strep Tag II标签可替换为其他常用标签,如GST、MBP等;融合蛋白变性条件还可使用6 M盐酸胍溶液,蛋白复性可使用透析、超滤溶液置换等方式,如变性条件组装的ExoBPmagbeads,可利用透析袋透析方式进行复性;可使用切向流离心、SEC、超滤、密度梯度离心等方式替换超速离心收集外泌体,外泌体来源可是组织体液、原代细胞培养液、细胞系培养液等。
本发明通过大量表达制备能够识别外泌体的融合蛋白,并将其固定于磁性粒子表面,利用其识别外泌体及磁性分离特性,无需超速离心机、层析系统等精密昂贵仪器,普通实验室及科研人员即可便捷经济富集外泌体,进行后续研究或应用。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (9)
1.一种识别外泌体的融合蛋白,其特征在于,包括支架蛋白和外泌体结合肽;所述支架蛋白包括hBD3蛋白和/或Syncytin-1蛋白N端;
所述hBD3蛋白和所述Syncytin-1蛋白N端由linker序列连接;
在所述支架蛋白的N末端、内部或C末端融合表达所述外泌体结合肽;
所述融合蛋白的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的识别外泌体的融合蛋白,其特征在于,所述外泌体结合肽包括ITGB1结合肽RGD4C、CD63结合肽CP05。
3.根据权利要求1或2所述的识别外泌体的融合蛋白,其特征在于,所述linker序列选自3(GS)linker、G4S linker、EA3K linker中的至少一种。
4.根据权利要求1所述的识别外泌体的融合蛋白,其特征在于,所述融合蛋白还包括蛋白分离标签;所述蛋白分离标签选自His、Strep Tag II、GST、MBP中的至少一种。
5.一种编码识别外泌体的融合蛋白的核苷酸,其特征在于,所述核苷酸的碱基序列如SEQ ID NO:2所示。
6.一种重组菌,其特征在于,所述重组菌表达权利要求1~4任一项所述的融合蛋白。
7.根据权利要求6所述的重组菌,其特征在于,所述重组菌为大肠杆菌。
8.一种细胞外泌体的富集方法,其特征在于,包括以下步骤:
(1)诱导权利要求6所述重组菌表达所述融合蛋白,分离所述融合蛋白;
(2)纯化并变性所述融合蛋白,得变性蛋白;
(3)加入磁珠固化所述变性蛋白;复性所述变性蛋白,得复性磁性粒子;
(4)收集细胞外泌体;将所述复性磁性粒子与所述细胞外泌体共孵育,通过磁性分离富集外泌体。
9.根据权利要求8所述的富集方法,其特征在于,在所述步骤(2)中,所述变性采用7 M~9 M尿素或5 M~7 M盐酸胍处理;在所述步骤(3)中,所述复性采用大量稀释复性法、透析、超滤溶液置换法中的至少一种;在所述步骤(4)中,所述收集细胞外泌体采用超速离心、切向流离心、SEC、超滤、密度梯度离心中的至少一种;所述细胞外泌体来自组织体液、原代细胞培养液、细胞系培养液中的至少一种。
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