CN108864308A - 一种mTAT-hEGF-kCD47融合蛋白及构建方法与应用 - Google Patents
一种mTAT-hEGF-kCD47融合蛋白及构建方法与应用 Download PDFInfo
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- CN108864308A CN108864308A CN201810826674.5A CN201810826674A CN108864308A CN 108864308 A CN108864308 A CN 108864308A CN 201810826674 A CN201810826674 A CN 201810826674A CN 108864308 A CN108864308 A CN 108864308A
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Abstract
本发明公开的一种mTAT‑hEGF‑kCD47融合蛋白,将mTAT、kCD47与hEGF融合表达,设计出mTAT‑hEGF‑kCD47的全基金组序列,人工合成后连接入pTrcHisB表达载体,构建成功mTAT‑hEGF‑kCD47重组表达质粒载体,经表达、纯化获得具有生物学活性的mTAT‑hEGF‑kCD47融合蛋白,具有低成本、高活性、作用时间久的优点。本发明mTAT‑hEGF‑kCD47融合蛋白不仅保留了TAT原有的穿透力和CD47的抑制非特异性免疫应答的作用,且对外源蛋白hEGF的活性几乎没有影响,使其在在制备促进表皮增殖的药物及护肤品中具有广阔的前景。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种mTAT-hEGF-kCD47融合蛋白,本发明还涉及一种mTAT-hEGF-kCD47融合蛋白的构建方法与应用。
背景技术
表皮生长因子(epidermal growth factor,EGF)具有促进表皮细胞增殖等多种功效。人表皮生长因子(human epidermal growth factor,hEGF)是一种由53个氨基酸残基组成的单链多肽,其分子量为6kDa,等电点pI为4.2。hEGF分子的稳定性较高,能耐受酸、碱、热以及胃蛋白酶和胰蛋白酶等的作用。大量研究证实hEGF具有刺激表皮细胞增殖和分化的能力,能通过促进表皮细胞内DNA的合成,加速新细胞取代老化细胞的进程,增加细胞对其他内源性生长因子的摄取,促进细胞分泌透明质酸和糖蛋白,从而恢复表皮的光滑和弹性、延缓皮肤衰老、减少皱纹,hEGF将在美容护肤方面具有十分重要的意义。此外,当表皮发生损害后,hEGF还可与细胞膜上的表皮生长因子受体(epidermal growth factor receptor,EGFR)特异性结合,启动EGF/EGFR信号转导通路,加速表皮细胞的增殖并刺激其分化成熟,从而修复受损皮肤。
天然的EGF稀少、难以穿过皮肤、易被免疫系统清除,从而限制了它的应用。天然hEGF资源很少,且产量低,因而价格昂贵;作为一种生物活性蛋白,溶液中的hEGF活性也只能维持1周左右,所以很难将其直接作为添加剂使用;更难以解决的是hEGF的生物利用度低,因为该分子本身并不具备有效穿过表皮的能力,而且还容易被皮肤内的免疫系统清除,这些问题都掣肘了hEGF的应用。如何解决这些问题和瓶颈,是EGF得以广泛使用的前提。
针对以上问题,目前的解决渠道有三种:一是采用基因工程技术可获得低价高产的hEGF;二是TAT蛋白转导技术;三是CD47具有减轻免疫系统的非特异性免疫应答反应的功能。
蛋白转导技术是近些年来运用比较广泛的一项技术,可使分子量超过100kD的蛋白质或其他大分子物质穿过细胞膜,甚至穿过血脑屏障和血脊屏障。研究证实一种来源于人类Ⅰ型免疫缺陷病毒(HIV-I)的转录反式激活因子(trans-activator oftranscription,TAT)的蛋白转导域(protein transduction domain,PTD),能够将分子量在15~120kD的不同生物大分子(如核酸、多肽、蛋白质等)高效、快速、安全地导入细胞,且导入后仍具有生物活性,并对宿主细胞几乎没有毒性。TAT蛋白转导域的发现为蛋白质治疗带来了新的曙光,显示出巨大的应用价值。目前,TAT蛋白转导技术已经成功将BDNF、NEP1-40、GDNF、Bcl-2、JIP等功能蛋白转导进入脑组织和神经元。TAT蛋白结构域修饰的蛋白质药物目前已经用于二期临床的试验。克里姆比尔神经医学中心的M.Tymianski博士将TAT-NR2B9C(C-terminal 9amino acids of N-methyl-d-aspartate receptors-2B)用于因脑动脉瘤而引发轻微中风的患者,结果显示90%以上的患者不会引起明显的神经性伤残。92位患者接受TAT-NR2B9C治疗,93位接受安慰剂治疗,没有发现TAT-NR2B9C引起的严重不良反应事件,表明TAT介导的蛋白质药物是安全的,这是人类首次将TAT修饰的蛋白药物用于临床研究,表明TAT作为介导蛋白质药物在临床的应用成为可能。近年来的研究表明,9个连续的Arg多肽同样具有蛋白转导的功能,且转导速度是TAT的9倍,将其命名为改良的TAT(modified TAT,mTAT),其序列为RRRRRRRRR。
CD47具有减轻免疫系统的非特异性免疫应答反应的功能。CD47又称整合素相关蛋白(integrin-associated protein,IAP)是一种广泛分布于多种细胞膜表面上的高度糖化的跨膜蛋白。CD47通过与其配体--信号调节蛋白α(SIRPα)形成CD47-SIRPα信号复合体,产生能降低巨噬细胞活性的抑制性信号,从而减轻免疫系统的非特异性免疫应答反应。CD47能抑制巨噬细胞活性的关键功能区GNYTSEVTELTREGETIIELK,将其命名为CD47关键结构域(key domain of CD47,kCD47),通过将kCD47和hEGF融合表达,达到抑制皮肤内免疫系统的非特异性免疫应答反应,从而有效维持hEGF的作用浓度。
基于上述分析,将mTAT、kCD47与hEGF融合表达,获得一个既具有蛋白转导功能又具有抗免疫清除能力的新的hEGF融合蛋白mTAT-hEGF-kCD47。
发明内容
本发明的第一个目的是提供一种mTAT-hEGF-kCD47融合蛋白,解决了大分子蛋白质不能主动穿透表皮、易被皮肤内的免疫系统清除的问题。
本发明的第二个目的是提供一种mTAT-hEGF-kCD47融合蛋白的构建方法。
本发明的第三个目的是提供一种mTAT-hEGF-kCD47融合蛋白的应用。
本发明所采用的第一个技术方案是,一种mTAT-hEGF-kCD47融合蛋白,mTAT-hEGF-kCD47融合蛋白的cDNA序列如序列1所示。
本发明的特征在于,
mTAT-hEGF-kCD47融合蛋白的蛋白质序列如序列2所示。
mTAT-hEGF-kCD47融合蛋白中,mTAT为具有连续的9个连续的Arg多肽,其氨基酸的序列RRRRRRRRR。
kCD47具体为CD47能抑制巨噬细胞活性的关键功能区GNYTSEVTELTREGETIIELK。
本发明所采用的第二个技术方案是,一种mTAT-hEGF-kCD47融合蛋白的构建方法,包括以下步骤:
步骤1,构建mTAT+hEGF+kCD47载体
取用GeneBank基因库中TAT、hEGF和CD47已知序列为基础,在序列的N端加上RRRRRRRRR,中间通过SGPG连接肽连接,模板序列设计为:mTAT+hEGF+kCD47;
采用载体pTrcHis,使用BamHI和XhoI双酶切穿梭pTrcHis质粒,得到载体大片段,将所述模板序列与载体大片段连接,得到重组穿梭质粒,测序正确后构建成功mTAT+hEGF+kCD47的表达载体,即得到pTrcHis-mTAT+hEGF+kCD47;
步骤2,mTAT+hEGF+kCD47蛋白表达及纯化
将构建成功的步骤1的pTrcHis-mTAT+hEGF+kCD47转化入大肠杆菌BL21菌株中,挑选3-5个阳性菌类克隆在氨苄抗性的LB培养基中培养,采用IPTG诱导蛋白;
将诱导后的全菌蛋白依次经过SDS-PAGE和考马斯亮蓝R250染色后,确定诱导表达条件和阳性菌落克隆,在37℃,0.5mM IPTG条件下表达mTAT+hEGF+kCD47融合蛋白;
将诱导表达mTAT+hEGF+kCD47融合蛋白的细菌经过超声破碎后,通过Ni离子交换柱亲和纯化mTAT+hEGF+kCD47融合蛋白,完成融合过程。
本发明的特征还在于,
步骤1中mTAT+hEGF+kCD47的表达载体的基因编码序列如序列表3所示。
步骤1中BamHI和XhoI双酶切切入点分别为:
BamHI酶切入点为序列表3中第1-6个核苷酸处,
XhoI酶切入点为序列表3中第286-291个核苷酸处。
本发明所采用的第三个技术方案是,一种mTAT-hEGF-kCD47融合蛋白在制备促进表皮增殖的药物及护肤品中的应用。
本发明的有益效果是:
(1)本发明mTAT-hEGF-kCD47融合蛋白,将mTAT、kCD47与hEGF融合表达,同时选用大肠杆菌偏好的密码子,在计算机辅助下设计出mTAT-hEGF-kCD47的全基金组序列,人工合成后连接入pTrcHisB表达载体,构建成功mTAT-hEGF-kCD47重组表达质粒载体,经表达、纯化获得具有生物学活性的mTAT-hEGF-kCD47融合蛋白,且具有低成本、高活性、作用时间久的优点;
(2)本发明mTAT-hEGF-kCD47融合蛋白,具有TAT可进行蛋白传送的特点,有效解决了大分子蛋白质不能主动穿透表皮的问题,同时也解决了该分子易被皮肤内的免疫系统清除的难题;将mTAT基因、CD47基因与外源蛋白hEGF连接,表达融合蛋白mTAT-hEGF-kCD47,不仅保留了TAT原有的穿透力和CD47的抑制非特异性免疫应答的作用,且对外源蛋白hEGF的活性几乎没有影响,使mTAT-hEGF-kCD47融合蛋白,在hEGF的应用中具有广阔的前景。
附图说明
图1是本发明一种mTAT-hEGF-kCD47融合蛋白的表达纯化示意图,
其中,泳道1表示蛋白maker,泳道2表示IPTG诱导前的mTAT-hEGF-kCD47菌液,泳道3表示IPTG诱导后的mTAT-hEGF-kCD47菌液,泳道4表示IPTG诱导后的mTAT-hEGF-kCD47细菌破碎上清液,泳道5表示IPTG诱导后的mTAT-hEGF-kCD47菌液破碎液沉淀,泳道6表示纯化后的mTAT-hEGF-kCD47重组蛋白;
图2是本发明一种mTAT-hEGF-kCD47融合蛋白促进表皮细胞增殖的结构示意图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
实验过程
一种mTAT-hEGF-kCD47融合蛋白的构建方法,包括以下步骤:
步骤1,构建mTAT+hEGF+kCD47载体
取用GeneBank基因库中TAT、hEGF和CD47已知序列为基础,在序列的N端加上RRRRRRRRR,中间通过SGPG连接肽连接,全序列委托金斯瑞生物公司合成。模板序列设计为:TAT+hEGF+kCD47;
采用载体pTrcHis,使用BamHI和XhoI双酶切穿梭pTrcHis质粒,得到载体大片段,将模板序列与载体大片段连接,得到重组穿梭质粒,测序正确后构建成功mTAT+hEGF+kCD47的表达载体,即得到pTrcHis-TAT+hEGF+kCD47,mTAT+hEGF+kCD47的表达载体的基因编码序列如序列表3所示。
其中BamHI和XhoI双酶切切入点分别为:BamHI酶切入点为序列表3中第1-6个核苷酸处,XhoI酶切入点为序列表3中第286-291个核苷酸处。
步骤2,mTAT+hEGF+kCD47蛋白表达及纯化
将构建成功的步骤1的pTrcHis-mTAT+hEGF+kCD47转化入大肠杆菌BL21(DE3)菌株中,挑选3-5个阳性菌类克隆在氨苄抗性的LB培养基中培养,采用IPTG诱导蛋白;
将诱导后的全菌蛋白依次经过SDS-PAGE和考马斯亮蓝R250染色后,确定诱导表达条件和阳性菌落克隆,在37℃,0.5mM IPTG条件下表达mTAT+hEGF+kCD47融合蛋白;
将诱导表达mTAT+hEGF+kCD47融合蛋白的细菌经过超声破碎后,通过Ni离子交换柱亲和纯化TAT+hEGF+kCD47融合蛋白,完成融合过程。
如图1所示,为mTAT-hEGF-kCD47融合蛋白的表达纯化示意图,其中,泳道1表示蛋白maker,泳道2表示IPTG诱导前的mTAT-hEGF-kCD47菌液,泳道3表示IPTG诱导后的mTAT-hEGF-kCD47菌液,泳道4表示IPTG诱导后的mTAT-hEGF-kCD47细菌破碎上清液,泳道5表示IPTG诱导后的mTAT-hEGF-kCD47菌液破碎液沉淀,泳道6表示纯化后的mTAT-hEGF-kCD47重组蛋白。
由上可知,得到的mTAT-hEGF-kCD47融合蛋白的cDNA序列如序列1所示;mTAT-hEGF-kCD47融合蛋白的蛋白质序列如序列2所示。将TAT与EGF融合表达,能穿透表皮,发挥EGF促新生表皮细胞合成,并应用于表皮的修复和更新;将kCD47和hEGF融合表达,达到抑制皮肤内免疫系统的非特异性免疫应答反应,延长融合蛋白半衰期,增加融合蛋白发挥功效的时间。
进行mTAT-hEGF-kCD47融合蛋白促进表皮生长的实验
将mTAT+hEGF+kCD47融合蛋白
将表皮细胞HSF(人皮肤成纤维细胞)和HSE(早代人皮肤上皮细胞)以10000个/孔种96孔板中,细胞贴壁后加入mTAT+hEGF+kCD47融合蛋白,作用浓度分别为0ng/ml、1ng/ml、10ng/ml、100ng/ml、1ug/ml、10ug/ml,每个浓度重复5次,作用24h后,用CCK-8测量细胞增殖情况,结果如图2所示:发现TmTAT+hEGF+kCD47融合蛋白可显著促进表皮细胞的增殖。
本发明mTAT-hEGF-kCD47融合蛋白不仅保留了TAT原有的穿透力和CD47的抑制非特异性免疫应答的作用,且对外源蛋白hEGF的活性几乎没有影响,使其在在制备促进表皮增殖的药物及护肤品中具有广阔的前景。
序列表
<110> 西安医学院
<120> 一种mTAT-hEGF-kCD47融合蛋白及构建方法与应用
<130> 无
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 277
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
cgccgtcgcc gccgccgtcg ccgtcgctcg ggtccgggta actcagattc cgaatgtccg 60
ctgtcgcacg atggctattg cctgcatgat ggcgtctgca tgtatattga agcgctggac 120
aaatacgcct gcaactgtgt ggttggctat atcggtgaac gttgtcagta ccgcgatctg 180
aaatggtggg aactgcgtag cggtccgggt ctcgagggta actacaccag cgaagtgacc 240
gaactgaccc gtgaaggcga aaccattatt gaactga 277
<210> 2
<211> 93
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 2
Arg Arg Arg Arg Arg Arg Arg Arg Arg Ser Gly Pro Gly Asn Ser Asp
1 5 10 15
Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val
20 25 30
Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val
35 40 45
Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu
50 55 60
Leu Arg Ser Gly Pro Gly Leu Glu Gly Asn Tyr Thr Ser Glu Val Thr
65 70 75 80
Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys
85 90
<210> 3
<211> 291
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
ggatcccgcc gtcgccgccg ccgtcgccgt cgctcgggtc cgggtaactc agattccgaa 60
tgtccgctgt cgcacgatgg ctattgcctg catgatggcg tctgcatgta tattgaagcg 120
ctggacaaat acgcctgcaa ctgtgtggtt ggctatatcg gtgaacgttg tcagtaccgc 180
gatctgaaat ggtgggaact gcgtagcggt ccgggtctcg agggtaacta caccagcgaa 240
gtgaccgaac tgacccgtga aggcgaaacc attattgaac tgaaactcga g 291
Claims (8)
1.一种mTAT-hEGF-kCD47融合蛋白,其特征在于,所述mTAT-hEGF-kCD47融合蛋白的cDNA序列如序列1所示。
2.根据权利要求1所述的一种mTAT-hEGF-kCD47融合蛋白,其特征在于,所述mTAT-hEGF-kCD47融合蛋白的蛋白质序列如序列2所示。
3.根据权利要求1或2所述的一种mTAT-hEGF-kCD47融合蛋白,其特征在于,所述mTAT-hEGF-kCD47融合蛋白中,mTAT为具有连续的9个连续的Arg多肽,其氨基酸的序列RRRRRRRRR。
4.根据权利要求1或2所述的一种mTAT-hEGF-kCD47融合蛋白,其特征在于,所述kCD47具体为CD47能抑制巨噬细胞活性的关键功能区GNYTSEVTELTREGETIIELK。
5.如权利要求1所述的一种mTAT-hEGF-kCD47融合蛋白的构建方法,其特征在于,包括以下步骤:
步骤1,构建mTAT+hEGF+kCD47载体
取用GeneBank基因库中TAT、hEGF和CD47已知序列为基础,在序列的N端加上RRRRRRRRR,中间通过SGPG连接肽连接,模板序列设计为:mTAT+hEGF+kCD47;
采用载体pTrcHis,使用BamHI和XhoI双酶切穿梭pTrcHis质粒,得到载体大片段,将所述模板序列与载体大片段连接,得到重组穿梭质粒,测序正确后构建成功mTAT+hEGF+kCD47的表达载体,即得到pTrcHis-mTAT+hEGF+kCD47;
步骤2,mTAT+hEGF+kCD47蛋白表达及纯化
将构建成功的步骤1的pTrcHis-mTAT+hEGF+kCD47转化入大肠杆菌BL21菌株中,挑选3-5个阳性菌类克隆在氨苄抗性的LB培养基中培养,采用IPTG诱导蛋白;
将诱导后的全菌蛋白依次经过SDS-PAGE和考马斯亮蓝R250染色后,确定诱导表达条件和阳性菌落克隆,在37℃,0.5mM IPTG条件下表达mTAT+hEGF+kCD47融合蛋白;
将诱导表达mTAT+hEGF+kCD47融合蛋白的细菌经过超声破碎后,通过Ni离子交换柱亲和纯化mTAT+hEGF+kCD47融合蛋白,完成融合过程。
6.根据权利要求5所述的一种mTAT-hEGF-kCD47融合蛋白的构建方法,其特征在于,所述步骤1中mTAT+hEGF+kCD47的表达载体的基因编码序列如序列表3所示。
7.根据权利要求6所述的一种mTAT-hEGF-kCD47融合蛋白的构建方法,其特征在于,所述步骤1中BamHI和XhoI双酶切切入点分别为:
BamHI酶切入点为序列表3中第1-6个核苷酸处,
XhoI酶切入点为序列表3中第286-291个核苷酸处。
8.如权利要求1所述的一种mTAT-hEGF-kCD47融合蛋白在制备促进表皮增殖的药物及护肤品中的应用。
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CN114437239A (zh) * | 2022-02-07 | 2022-05-06 | 西安医学院 | 一种重组融合蛋白TAT-mCNTF-CD47及其制备方法和用途 |
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