CN116217693A - 一种芋螺毒素外泌体及其应用 - Google Patents
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Abstract
本发明公开了一种芋螺毒素外泌体及其应用。芋螺毒素CONOT‑25的氨基酸序列如SE Q ID NO.1所示。本发明从芋螺基因组中分离得到了一个新型芋螺毒素,且提供了一种效率更高,提取外泌体纯度更高的技术。同时,临床试验显示,新型芋螺毒素的外泌体具有较好的除皱效果,显示了在美容除皱领域中的巨大应用前景。
Description
技术领域
本发明属于分子生物和细胞生物技术领域,具体涉及一种芋螺毒素外泌体及其应用。
背景技术
芋螺(Cone Snail),又名“鸡心螺,”主要生长于热带海域,属软体动物门,腹足纲,芋螺科,是一种利用毒素捕食猎物的肉食性动物。芋螺具有相当丰富的种类,目前,全球已知的芋螺种类约有900多种,而在我国发现的芋螺种类约有100多种,主要分布在广东、广西、西沙群岛、海南岛及台湾海域。
芋螺毒素(conotoxin)是由芋螺毒液管和毒囊内壁的毒腺分泌得到的,每种芋螺的毒液中含有50-200个活性多肽,芋螺毒素具有很强的特异性,因此,不同的芋螺所含的芋螺毒素种类各有不同。据现有资料显示,每一种芋螺都有超过100种不同的芋螺毒素,因此理论上自然界中会有超过90000种天然的芋螺毒素,这是一个十分庞大的自然资源库,具有十分广阔的研究前景。芋螺毒素大多数由12~40个氨基酸组成,富含二硫键,包含了迄今为止最小的神经毒素。每个芋螺毒素均由单一的mRNA编码,其原始翻译产物是一种特定的前蛋白原前体化合物,由高度保守的信号肽序列、中间前体段和成熟毒素段三部分组成。根据芋螺毒素二硫键框架和高度保守的信号肽序列,可以将芋螺毒素分为若干个超家族,如A,M,O,P,I,S,T,I,V,Y,J等14个超家族,根据同一超家族中作用靶位的不同又可以将芋螺毒素进行进一步细分,如分为α、ω、μ、δ、κ、ψ等类型。
而外泌体(exosome)是由细胞分泌的纳米级球形脂质双层外囊泡,携带核酸、蛋白质、脂质等生物活性物质,可作为细胞间通讯和物质交换的介质在机体的生理和病理过程中发挥作用。外泌体蛋白组分主要分为两类,一类是公共组分,它们参与囊泡形成和分泌的过程,即外泌体无处不在,包括膜转运和融合相关蛋白,热休克蛋白,四跨膜蛋白超家族等;另一类是特异性组分,其与其前体细胞密切相关,即具有细胞特异性,如来自抗原呈递细胞的CD45和MHC-II。与其他合成载体相比,外泌体的内源性和异质性使其在疾病诊断和治疗领域具有广泛而独特的优势。然而,外泌体的储存稳定性低、低回收率、低纯度和弱靶向性限制了其临床应用。
发明内容:
本发明从芋螺基因组和多肽组中分离鉴定了一个91个氨基酸的多肽,具有XXII半胱氨酸框架,属E超家族,是一种新型芋螺毒素,命名为CONOT-25。
本发明的第一个目的是提供一种新型芋螺毒素CONOT-25,其氨基酸序列为:Met-Met-Thr-Arg-Val-Phe-Val-Met-Phe-Phe-Leu-Leu-Val-Leu-Thr-Glu-Gly-Trp-Pro-Arg-Leu-Thr-Asp-Ser-Asp-Cys-Arg-Arg-Gly-Pro-Asn-Met-His-Ile-Thr-Cys-Phe-Lys-Asp-Gln-Ser-Cys-Gly-Leu-Ile-Val-Lys-Arg-Asn-Gly-Arg-Leu-Ser-Cys-Thr-Leu-Asn-Cys-Lys-Cys-Arg-Arg-Asn-Glu-Ser-Cys-Leu-Pr o-Ser-Glu-Glu-Val-Asp-Trp-Asp-His-Arg-Asp-Met-Lys-Ile-Val-Ile-Cys-Pro-Lys-Pro-Cys-Gln-Glu(SEQ ID NO.1)。
本发明的第二个目的是提供一种上述的芋螺毒素CONOT-25的编码基因。
本发明的第三个目的是提供一种重组表达载体,含有上述的编码基因。
优选,所述重组表达载体的原始表达载体为pc-DNA5/FRT/TO。
本发明的第四个目的是提供一种基因工程细胞,其为整合有上述的编码基因或转化有上述的重组表达载体的细胞。
优选,所述的细胞为Flp-In-CHO和/或POR-Flp-In-CHO细胞。
本发明的第五个目的是提供一种上述细胞所分泌的外泌体。
本发明的第六个目的是提供上述的芋螺毒素CONOT-25或外泌体在制备美容除皱产品,例如化妆品或药物中的应用。
与现有技术相比,本发明具有以下有益效果:本发明从芋螺基因组中分离得到了一个新型芋螺毒素,且提供了一种效率更高,提取外泌体纯度更高的技术。同时,临床试验显示,新型芋螺毒素的外泌体具有较好的除皱效果,显示了在美容除皱领域中的巨大应用前景。
附图说明
图1是纳米颗粒跟踪分析测定芋螺毒素-外泌体的粒径效果。
图2是芋螺毒素-外泌体的累积渗透。
图3是皱纹体积变化。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1芋螺毒素CONOT-25序列的鉴定
剪取芋螺的毒管部分并匀浆,利用5%乙酸提取,冷冻离心得到粗毒。随后利用HPLC对粗毒进行分离纯化,随后对提取出的蛋白进行活性和序列测定,得到芋螺毒素CONOT-25,其氨基酸序列为:Met-Met-Thr-Arg-Val-Phe-Val-Met-Phe-Phe-Leu-Leu-Val-Leu-Thr-Glu-Gly-Tr p-Pro-Arg-Leu-Thr-Asp-Ser-Asp-Cys-Arg-Arg-Gly-Pro-Asn-Met-His-Ile-Thr-Cys-Phe-Lys-Asp-Gln-Ser-Cys-Gly-Leu-Ile-Val-Lys-Arg-Asn-Gly-Arg-Leu-Ser-Cys-Thr-Leu-Asn-Cys-Lys-Cys-Arg-Arg-Asn-Glu-Ser-Cys-Leu-Pro-Ser-Glu-Glu-Val-Asp-Trp-Asp-His-Arg-Asp-Met-Lys-Ile-Val-Ile-Cys-Pro-Lys-Pro-Cys-Gln-Glu(SEQ ID NO.1),具有XXII型的半胱氨酸框架,属于E类超家族,命名为芋螺毒素CONOT-25,编码基因命名为CT25基因。
实施例2表达载体细胞系的建立
从液氮中取出细胞冻存管快速浸入37℃温水中,快速晃动,使其在1~2min内快速解冻。之后将其加入含有10%牛血清和1%青霉素-链霉素的DEME细胞培养基(Gibco)的15mL离心管中,1200rpm离心3min。弃上清,加入1mL含10%胎牛血清的Ham’s F12完全培养基,对细胞沉淀进行吹吸混匀,吹洗3~5次左右。准备一个细胞培养皿,加入10mL左右的完全培养基,取1mL重悬液加入到培养皿中,轻轻混匀后放入37℃,5% CO2的恒温培养箱中培养24h,第二天观察细胞贴壁情况。当细胞达到80%汇合度时,胰蛋白酶消化传代,传代比例为1:3,计为第1代细胞,取生长良好的第3-6代细胞进行实验。
实施例3:芋螺毒素表达载体构建
以芋螺毒素CONOT-25为目的序列,pc-DNA5/FRT/TO为表达载体,进行芋螺毒素CONOT-25表达载体构建。
(1)用EcoRⅠ和EcoRⅤ双酶切表达载体pc-DNA5/FRT/T同时在目的CT25基因序列(SEQ ID NO.2所示)两侧加入EcoRⅠ和EcoRⅤ酶切位点,胶回收载体片段和CT25基因片段,得到纯化后的片段并用T4连接酶连接。通过E.coli DH5α感受态转化连接产物,涂布至含有氨苄抗生素的LB平板中进行阳性筛选。
(2)根据目的序列,利用Primer 5.0软件设计引物。挑选平板上的单菌落并扩大培养,利用菌落PCR对其进行鉴定。扩增产物经凝胶电泳分离后进行观察和照相,将电泳条带大小符合要求的PCR产物送去生物公司进行测序,确认芋螺毒素CONOT-25的编码基因CT25基因插入到pc-DNA5/FRT/TO表达载体中,获得重组表达质粒。
实施例4:新型芋螺毒素细胞表达系的构建和筛选
(1)细胞系构建
取上述经过处理后且处于对数生长期的Flp-In-CHO和POR-Flp-In-CHO细胞,用完全培养基将细胞稀释至1.5×108个·L-1,以2mL/孔接种于6孔板中,置于37℃、5%CO2恒温培养箱中培养24h。待细胞长成70%单层时弃去培养液,用无血清培养基洗涤细胞2次,利用LipofectamineTM 2000转染液(8μL)将上述鉴定正确的重组表达质粒(0.25μg)和编码Flp重组酶的辅助质粒pOG44(2.25μg,NTCC典型培养物保藏中心)转染至细胞。以不含目的基因序列的pcDNA5/FRT/TO空载体质粒作为阴性对照,按照上述方法共转染至细胞。转染48h后,向培养基中加终浓度为500mg·L-1的潮霉素B进行阳性克隆的筛选。由此得到转染有重组表达质粒的Flp-In-CHO和POR-Flp-In-CHO细胞以及转染有空载体的Flp-In-CHO和POR-Flp-In-CHO细胞。
(2)qRT-PCR检测
利用TRIzol溶液将细胞充分裂解,提取细胞总RNA并逆转录成cDNA。利用MonAmpT MChemoHS Specifity Plus qPCR Mix试剂盒进行实时定量荧光PCR检测,以GADPH为内参,通过2-ΔΔCt法进行数据分析。
实施例5:新型芋螺毒素-外泌体的制备
采取将上清液先超滤浓缩再超离沉淀的方式,提高外泌体的提取效率和浓度。
(1)收集细胞上清液
将上述转染完成后的Flp-In-CHO和POR-Flp-In-CHO细胞继续进行传代,计转染完成后的细胞为1代细胞,收集足够多的生长良好的第3-6代细胞培养的上清液。
(2)超滤浓缩细胞上清液
向100kD的超滤离心管中加入上述上清液1200mL,以4000×g转速离心30min,加入pH 7.4的1×PBS,轻柔吹打,得到含外泌体的超滤浓缩液约120mL。
(3)密度梯度离心浓缩液
将上述超滤浓缩液按照300×g离心10min,2000×g离心10min,10000×g离心30min,100000×g离心70min×2次的步骤进行梯度离心,此时管底中淡黄沉淀物即为我们所需的外泌体,用1mL 1×PBS轻柔充分地吹打混匀,使外泌体充分重悬,得到外泌体悬液,保存于-80℃冰箱内备用。
实施例6:新型芋螺毒素-外泌体的粒径效果
本次实验采用了lzon公司的qNano纳米粒子分析系统进行外泌体的粒径计量分析,然后应用微流式成像系统对外泌体进行荧光标记显示,最后利用电子显微镜直接观察到外泌体的形态大小。纳米粒子分析系统的原理是运用可调式纳米孔薄膜和电阻脉冲感应器检测外泌体悬液中各个颗粒物的粒径大小,仪器可测定的直径范围为50~10000nm,可从粒径大小上确认是否是外泌体。操作方法较为简单,可将收集到的外泌体悬液用上样缓冲液按照1:1的比例稀释后直接上机进行观察。
具体粒径如图1所述,外泌体粒径分布峰值为(108.5±9.1)nm,浓度为8.125×1010粒子/mL。
实施例7:新型芋螺毒素-外泌体的渗透效果评估
(1)皮肤样品处理
将健康状况良好的大鼠的腹部绒毛用脱毛膏去除干净,然后将大鼠颈椎脱臼处死,小心迅速剥皮,将取下的腹部皮肤平铺于干净的玻璃板上,角质层朝下。用刀片小心剔除皮下的脂肪组织及粘连物,用生理盐水反复冲洗干净,后置于生理盐水中,并放置4℃冰箱中保存备用。每次实验前查看皮肤的完整性,不能有任何破损。
(2)渗透效果评估实验
采用水平扩散池,即由两只筒状玻璃管对合而成,鼠皮置于两者之间。两只玻璃管为两室,分别为扩散池和接收池。在扩散池和接收池中分别放入磁力搅拌子以维持接收池的动态环境,在接收池的右边连一取样管,供进样、取样及排除气泡用。接收池的体积为5mL,扩散面积为2.8cm2。从冰箱中取出离体鼠皮,以生理盐水洗净,用滤纸吸干表面水分,将处理好的鼠皮固定于扩散池和接收池之间,角质层朝向扩散池。然后向扩散池中加入上述的外泌体悬液5mL,接收池中加入生理盐水5mL,水域恒温至32℃,恒速搅拌(200r/min)。分别于实验开始后2、4、8、12、24h,从样品接收池中取出1mL作为样品液,同时,再补加同样体积的32℃预热的生理盐水,排除气泡。将取出的样品液经0.45μm的微孔滤膜过滤后,进行含量鉴定。所测样品峰面积根据当日标准曲线计算浓度,然后按照下列公式计算单位面积累积透过量:
其中Cn为第n次取样测得的药物质量浓度(μg/mL),Ci为第i次取样测得的药物质量浓度(μg/mL),Vi为取样体积,A为扩散池有效渗透面积(cm2),V为扩散池容积(mL),以时间t为横坐标,Q为纵坐标,绘制经皮渗透曲线。
累积透过量的结果如图2所示,随着时间的增长,单位面积的累积渗透量逐渐升高,24h后达到18.459μg。
实施例8:新型芋螺毒素-外泌体的去皱效果评估
配制含有芋螺毒素外泌体的乳液,按照以下浓度配制含有芋螺毒素外泌体的乳液:实施例5的芋螺毒素外泌体250ppm,甘露糖5%(w/v),海藻糖1%(w/v)。配置方法:将甘露糖和海藻糖溶解在水中,高温灭菌,待冷却至室温,加入芋螺毒素外泌体。
本发明所使用表征新型芋螺毒素外泌体可用于皮肤去除皱纹的方法是临床测试,一共选择了30名女性志愿者参与测试。志愿者年龄在35-55岁之间。测试时将护肤品或者化妆品0.1克均匀涂抹于志愿者眼角部位。分别在使用前,使用后1小时测试志愿者皱纹体积。测试所使用设备为PRIMOS CR皱纹分析仪。
将每一位志愿者在使用后15分钟,使用后1小时测试得到的皱纹体积减去测试前皱纹体积,获得该志愿者在不同时间点上皱纹减少的值。将此减少值除以测试前皱纹体积,获得该志愿者的皱纹减少百分比(图3)。将所有志愿者的皱纹体积减少百分比做数学平均,获得本次测试的皱纹体积减少平均值。结果如图3所示,由结果可以看出,含有新型芋螺毒素外泌体的产品均表现出了良好的祛皱效果。添加了新型芋螺毒素(芋螺毒素CONOT-25)外泌体的测试结果(每组柱子的右侧)为皱纹减轻了5.6%,21.9%,17.0%,11.4%和19.6%。而未添加新型芋螺毒素外泌体的对照组(每组柱子的左侧),其效果显著差于实验组(Treatment)。芋螺毒素CONOT-25的蛋白序列如SEQ ID NO.1所示,具体如下:
MMTRVFLVMFFLLVLTEGWPRLTDSDCRRGPNMHITCFKDQSCGLIVKRNGRLSCTLNCKCRRNESCLPSEEVDWDHRDM
KIVICPKPCQE
芋螺毒素CONOT-25的编码基因—CT25基因的核酸序列如SEQ ID NO.2所示,具体如下:ATGATGACACGTGTGTTCCTTGTGATGTTCTTCCTCTTGGTGCTAACAGAGGGATGGCCACGTCTGACTGATTCGGACTGTCGGAGGGGGCCTAATATGCATATCACGTGCTTTAAAGATCAGTCATGTGGACTGATTGTAAAGCGGAACGGAAGATTGAGCTGCACACTGAACTGTAAGTGTAGACGTAACGAAAGCTGTCTTCCGAGTGAAGAAGTCGACTGGGATCACAGGGATATGAAAATAGTCATCTGCCCGAAACCCTGTCAAGAA。
Claims (9)
1.一种新型芋螺毒素CONOT-25,其特征在于,氨基酸序列为:Met-Met-Thr-Arg-Val-Phe-Val-Met-Phe-Phe-Leu-Leu-Val-Leu-Thr-Glu-Gly-Trp-Pro-Arg-Leu-Thr-Asp-Ser-Asp-Cys-Arg-Arg-Gly-Pro-Asn-Met-His-Ile-Thr-Cys-Phe-Lys-Asp-Gln-Ser-Cys-Gly-Leu-Ile-Val-Lys-Arg-Asn-Gly-Arg-Leu-Ser-Cys-Thr-Leu-Asn-Cys-Lys-Cys-Arg-Arg-Asn-Glu-Ser-Cys-Leu-Pro-Ser-Glu-Glu-Val-Asp-Trp-Asp-His-Arg-Asp-Met-Lys-Ile-Val-Ile-Cys-Pro-Lys-Pro-Cys-Gln-Glu。
2.一种权利要求1所述的芋螺毒素CONOT-25的编码基因。
3.根据权利要求2所述的编码基因,其特征在于,其核苷酸序列如SEQ ID NO.2所示。
4.一种重组表达载体,其特征在于,含有权利要求2或3所述的编码基因。
5.根据权利要求4所述的重组表达载体,其特征在于,所述重组表达载体的原始表达载体为pc-DNA5/FRT/TO。
6.一种基因工程细胞,其特征在于,为整合有权利要求2或3所述的编码基因或转化有权利要求4或5所述的重组表达载体的细胞。
7.根据权利要求6所述的基因工程细胞,其特征在于,所述的细胞为Flp-In-CHO和/或POR-Flp-In-CHO细胞。
8.一种权利要求6或7所述的细胞所分泌的外泌体。
9.权利要求1所述的芋螺毒素CONOT-25或权利要求8所述的外泌体在制备美容除皱的产品中的应用。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1765922A (zh) * | 2004-10-27 | 2006-05-03 | 戚正武 | T超家族芋螺毒素肽 |
US20170157016A1 (en) * | 2012-04-13 | 2017-06-08 | Activen | Cosmetic composition comprising a muconopeptide |
CN108587998A (zh) * | 2018-05-17 | 2018-09-28 | 浙江大学 | 一种外泌体、外泌体的制备方法及其在制备皮肤浅表性肿瘤的药物中的应用 |
CN113797118A (zh) * | 2021-11-01 | 2021-12-17 | 临沂大学 | 一种包载芋螺毒素的壳聚糖纳米粒乳液及其制备方法 |
CN113952286A (zh) * | 2021-11-01 | 2022-01-21 | 广州远想医学生物技术有限公司 | 一种靶向酪氨酸酶抑制剂、制备方法和应用及美白霜 |
CN114106202A (zh) * | 2021-11-25 | 2022-03-01 | 西安德诺海思医疗科技有限公司 | 一种芋螺毒素和胶原蛋白融合蛋白及其制备方法和应用 |
-
2023
- 2023-01-06 CN CN202310020990.4A patent/CN116217693B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1765922A (zh) * | 2004-10-27 | 2006-05-03 | 戚正武 | T超家族芋螺毒素肽 |
US20170157016A1 (en) * | 2012-04-13 | 2017-06-08 | Activen | Cosmetic composition comprising a muconopeptide |
CN108587998A (zh) * | 2018-05-17 | 2018-09-28 | 浙江大学 | 一种外泌体、外泌体的制备方法及其在制备皮肤浅表性肿瘤的药物中的应用 |
CN113797118A (zh) * | 2021-11-01 | 2021-12-17 | 临沂大学 | 一种包载芋螺毒素的壳聚糖纳米粒乳液及其制备方法 |
CN113952286A (zh) * | 2021-11-01 | 2022-01-21 | 广州远想医学生物技术有限公司 | 一种靶向酪氨酸酶抑制剂、制备方法和应用及美白霜 |
CN114106202A (zh) * | 2021-11-25 | 2022-03-01 | 西安德诺海思医疗科技有限公司 | 一种芋螺毒素和胶原蛋白融合蛋白及其制备方法和应用 |
Non-Patent Citations (3)
Title |
---|
ABALDE, S.等: "Conus ammiralis clone P_042 conotoxin precursor superfamily E mRNA, partial cds", 《GENBANK》, vol. 1, pages 110 - 111 * |
TIANQI LIU等: "Highly efficient conotoxin delivery enabled by a bio-derived ionic liquid", 《JOURNAL OF MOLECULAR LIQUIDS》, vol. 367, pages 1 - 11 * |
孟海玲等: "新芋螺毒素基因的克隆及其遗传进化分析", 《生命科学研究》, vol. 19, no. 02, pages 106 - 113 * |
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