CN106519040A - 一种含肿瘤坏死因子相关凋亡诱导配体的融合蛋白和其制备方法及由该蛋白自组装的纳米粒 - Google Patents
一种含肿瘤坏死因子相关凋亡诱导配体的融合蛋白和其制备方法及由该蛋白自组装的纳米粒 Download PDFInfo
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Abstract
本发明属生物制药领域,具体涉及一种TRAIL变体蛋白表达、纯化,自组装。该重组蛋白由含有TRAIL融合蛋白基因的质粒在大肠杆菌表达,产生活性很高的该重组蛋白。该重组蛋白采用离心的方式进行简单、快速地纯化。纯化的蛋白在生理条件下自组装成为200纳米的微粒。静脉注射肿瘤移植小鼠后,该重组蛋白有效地在小鼠的肿瘤部位聚集。该重组蛋白腹腔注射肿瘤移植小鼠,有效抑制肿瘤生长。经简单工艺纯化后的该重组蛋白,用于肿瘤治疗。
Description
一、技术领域
本发明属生物制药领域,具体涉及一种TRAIL变体蛋白制备及其应用。
二、背景技术
肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)是TNF超家族一个新成员,它与肿瘤细胞上的死亡受体4、5(death receptor 4、5,DR4,DR5)结合后,形成诱导死亡信号复合物(death-inducing signaling complex,DISC),DISC结合Caspase8前体,激活Caspase8前体并水解Caspase8前体,进而激活caspases-3,-6与-7,从而引起细胞凋亡。由于TRAIL可以选择性地诱导黑色素癌,淋巴癌,结肠癌,肺癌及乳腺癌等多种恶性肿瘤细胞凋亡但是对大部分正常细胞的影响很小,因而TRAIL是一个非常有前景的抗肿瘤药物(Ichikawa et al.,Cancer Res,2001;Frese et al.,Nat Med,2006)。
为了增强TRAIL诱导肿瘤细胞凋亡活性,一些研究者做了不少有益尝试。Rozanov等研究表明TRAIL的N端与亮氨酸融合表达,可以稳定TRAIL的三聚体结构,进而提高TRAIL诱导凋亡活性(Rozanov et al.,Mol Cancer Ther,.2009)。Qin将ELP与His tag融合后,结果也显著提高TRAIL诱导凋亡活性(Qin et al.,Nat Med,2001)。之前,我们的研究结果表明,RGD与TRAIL(RGD-TRAIL)进行融合表达后,可以提高对于一些含有ανβ3和ανβ5整联蛋白细胞亲和力,在体内结果也显示有更多RGD-TRAIL融合蛋白进入肿瘤组织。RGD-TRAIL存在5个半胱氨酸或两对二硫键,若在E.coli进行表达纯化,很难获得可溶性的RGD-TRAIL。此外,RGD-TRAIL粒径较小(8nm),在体内半衰较短,会影响其在体内的抗肿瘤效果。
因此,提高RGD-TRAIL可溶性表达,快速且简单纯化蛋白,提高RGD-TRAIL诱导肿瘤细胞凋亡活性及有效放大RGD-TRAIL蛋白粒径,是本发明要解决的问题。
三、发明内容
本发明解决的问题是提高RGD-TRAIL可溶性表达,简化蛋白纯化工艺,增强其诱导肿瘤细胞凋亡活性及改善药物代谢动力学,以取得在体内有良好的抗肿瘤效果。
为了解决此问题,本发明是将RGD-TRAIL与类弹性蛋白多肽(ELP)进行融合表达,来实现融合蛋白可溶性表达,利用ELP嵌段具有自聚集性质,通过可逆相变方式纯化蛋白,以实现简单快速纯化蛋白及RGD-TRAIL-ELP自组装成纳米微粒,达到放大蛋白径粒目的。所述的类弹性蛋白多肽其氨基酸序列是:
GHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVP。
所述融合蛋白RGD-TRAIL-ELP的序列:
VRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGGGACDCRGDCFCGGPDGHGVGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVP
所述的可逆相变纯化方式是:细菌破碎液4度12000g离心5分钟取上清;将上清在40度下加热,40度下12000g离心取5分钟,取沉淀部分;将沉淀部分用预冷的pbs缓冲液溶解,放在4度下15分钟,4度12000g离心5分钟取上清,如此4度离心取上清及40度下取沉淀循环三次;所述的自组装方法是:纯化后的RGD-TRAIL-ELP经冷冻干燥的,用生理盐水溶解(蛋白质的终浓度为0.5mg/ml)。
与现有的TRAIL变体相比,本发明的特色和创新之处在于:
(1)发明了一种融合类弹性类多肽的RGD-TRAIL变体蛋白,实现了可溶性表达,并通过离心的方法纯化了该蛋白。该蛋白可以形成较多的三聚体形式,进而提高其诱导肿瘤细胞凋亡活性。
(2)发明了一种RGD-TRAIL变体蛋白可以在生理条件下自组装成纳米微粒,并在肿瘤动物模型等证实该蛋白在体内能产生更好地肿瘤抑制效果。
综上所述,本发明提供了一种全新的、高效的、安全的、低廉的、RGD-TRAIL变体蛋白制备方法,该蛋白用于肿瘤抑制。
四、附图说明
图1、融合类弹性类多肽的RGD-TRAIL变体蛋白(RGD-TRAIL-ELP)表达与纯化
(1A)SDS-PAGE分析RGD-TRAIL-ELP表达:泳道1:细菌未诱导;泳道2:细菌用IPTG诱导后离心破碎后上清部分;泳道3:第一轮ITC后可溶性蛋白;泳道4:第二轮ITC后可溶性蛋白;泳道5:第三轮ITC后可溶性蛋白;泳道6:Marker。非变性(-DTT)聚丙烯酰胺凝胶电泳(1B)及HPLC(1C)分析RGD-TRAIL-ELP。
图2、RGD-TRAIL-ELP粒径表征
动态光散射测定0.5mg/ml RGD-TRAIL-ELP在10℃与37℃下粒径分布(2A),冷冻蚀刻-透射电镜测定RGD-TRAIL粒径(37℃,2B)及RGD-TRAIL-ELP粒径(10℃,2C)及(37℃,2D);37℃及10℃ RGD-TRAIL-ELP溶液(2E)。
图3、细胞流式仪检测RGD-TRAIL及RGD-TRAIL-ELP浓度与COLO-205细胞凋亡数量关系:(3A)重组蛋白浓度与COLO-205细胞凋亡率关系图;(3B)不同浓度下的重组蛋白诱导COLO-205细胞凋亡代表性结果。
图4、重组蛋白在COLO-205肿瘤模型鼠中的各个组织中分布情况(4A),定量分析给药24小时后各个组织中荧光强度(4B与4C)。
图5、RGD-TRAIL及RGD-TRAIL-ELP给药后,小鼠肿瘤体积变化图
五、具体实施方式
实施例1 RGD-TRAIL-ELP质粒的构建
ELP[VH4-5]基因序列为:5’-CCACGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTCACGGCGTACCGGGCCACGGTGTTCCTGGTCACGGCGTGCCGGGCTGGC-3(其氨基酸序列为VPGVG-VPGHG-VPGVG-VPGHG-VPGHG),由南京金斯瑞公司合成,ELP[VH4-40](8个ELP[VH4-5]序列重复)由ELP[VH4-5]通过RDL方法延长;ELP[VH4-40]通过BamHI及HindIII与带有RGD-TRAIL基因片段pET-23a进行连接。所有目的基因序列用测序进行鉴定(T7及T7-ter)。经测序鉴定,RGD-TRAIL-ELP基因已经插入到pET-23a中的NdeI与HindIII中。
实施例2 RGD-TRAIL-ELP表达纯化及鉴定
带有RGD-TRAIL-ELP质粒转入BL21(DE3)后,构建工程菌,培养于4升TB培养基,当OD值达到0.6值,加IPTG诱导,于30℃过夜诱导,收集菌体,用超声破碎仪进行细胞破碎,细菌破碎液4度12000g离心5分钟取上清;将上清在40度下加热,40度下12000g离心取5分钟,取沉淀部分;将沉淀部分用预冷的pbs缓冲液溶解,放在4度下15分钟,4度12000g离心5分钟取上清,如此4度离心取上清及40度下取沉淀循环三次。
用SDS-PAGE鉴定RGD-TRAIL-ELP在E.coli可溶性表达情况及分析每轮ITC纯化后蛋白纯度。用非还原性的PAGE胶(-DTT)鉴定RGD-TRAIL-ELP多聚体含量。
结果如附件图1所示:RGD-TRAIL-ELP在E.coli以可溶的形式进行表达,经ITC纯化后,得率为10mg/L。纯化后的RGD-TRAIL-ELP[V5-40]用变性还原的聚丙烯酰胺凝胶电泳分析。GD-TRAIL-ELP[V5-40]在变性还原条件下均为一条带,分子量在37kDa左右,与理论分子量接近。非变性胶的结果表明RGD-TRAIL-ELP[V5-40]存在三条带。其分子量分别为37,78,与120kDa。经初步计算,RGD-TRAIL-ELP[V5-40]三聚体占总量的38%。
实施例3 融合蛋白凋亡诱导效应的检测
用人结肠癌细胞(Human COLO rectal carcinoma cells,COLO205 cells)检测所纯化出来的RGD-TRAIL-ELP活性。COLO205培养于含有10%牛血清RPMI 1640。细胞(105)经过一系列浓度梯度的RGD-TRAIL或RGD-TRAIL-ELP诱导处理,经胰酶消化后从培养孔中吸出,PBS洗两遍,300g离心力离心5分钟,弃上清,然后用300μL结合缓冲液重悬,加入终浓度为2μg/mL的Annexin V-FITC室温孵育,10分钟后将细胞转移到流式管子,每管加入1μg的碘化吡啶(PI)后,细胞在30分钟内用流式细胞仪分析。
结果如附件图3所示,RGD-TRAIL与RGD-TRAIL-ELP半致量(EC50)分别为1与0.35nM(20.57与13.61ng/ml),因而RGD-TRAIL-ELP诱导凋亡活性可提高3倍。
实施例4 融合蛋白体内肿瘤靶向效应的检测
5~6周龄雌性裸鼠背部右上侧皮下接种106个COLO-205结肠癌细胞。当肿瘤达到300~500mm3时,随机分组,将用Cy5.5标记好的蛋白尾静脉注射小鼠5mg/kg(100μg),每组三只。尾静脉注射24小时后,处死小鼠,取出脏器及肿瘤组织,后用PBS清洗三次,置于黑色的板上,用小动物成像仪成像。为检测各个器官的荧光分布,用小动物成像仪所内置的分析软件对小鼠的脏器及肿瘤组织进行荧光半定量分析。
结果如附件图4所示,RGD-TRAIL与ELP给药组中,其肿瘤组织只检测出微弱的荧光,但在肾脏与肝脏组织中存在较强荧光信号。而RGD-TRAIL-ELP给药组中,有强荧光在肿瘤部位富集,而其荧光强度相当于2.5倍RGD-TRAIL给药组中的肿瘤组织,在其它组织中的荧光较弱。
实施例5 融合蛋白体内肿瘤抑制效应的检测
5~6周龄雌性裸鼠皮下接种2.5×106的COLO-205结肠癌细胞。当肿瘤达到~100mm3时,随机分组。RGD-TRAIL-ELP在给药之前,加热至37℃以形成纳米粒。RGD-TRAIL或者RGD-TRAIL-ELP(500μg/mL)组分别给药1.5及4.5nmol;在联合给药组中,ELP及RGD-TRAIL每种蛋白均注射4.5nmol。包括PBS组在内,共有6组的治疗组,每组5只,而给药方式均为腹腔注射,每隔两天给药一次,共注射14天。肿瘤的大小用电子游标卡尺进行测量,肿瘤的体积计算参照下面的公式:V=S2×L×0.5,其中V代表肿瘤体积,S代表肿瘤的短径,L代表肿瘤的长径。
结果如附件图5所示,在COLO-205肿瘤模型鼠中,30μg(1.4nmol)的RGD-TRAIL低剂量给药后,同PBS相比,肿瘤生长只受到轻微抑制,而等摩尔数的RGD-TRAIL-ELP(1.4nmol,55μg)却能显著地抑制肿瘤生长,且比高剂量的RGD-TRAIL(90μg,4.2nmol)肿瘤抑制效果更明显。用ELP与RGD-TRAIL进行共注射,其产生的治疗治疗效果与单独用RGD-TRAIL给药差异不是很显著。尤其是当用RGD-TRAIL-ELP进行高剂量(4.3nmol,165μg)给药10天后,小鼠的肿瘤逐渐消退。
本发明实施例是将RGD-TRAIL与ELP进行融合并且重组表达,其重组蛋白RGD-TRAIL-ELP在E.coli以可溶的形式进行表达,经可逆相变纯化后,得率为10mg/L。纯化后的RGD-TRAIL-ELP用变性还原的聚丙烯酰胺凝胶电泳分析,图1A结果显示RGD-TRAIL-ELP在变性还原条件下均为一条带,分子量在37kDa左右,与理论分子量接近。非变性胶的结果表明RGD-TRAIL-ELP存在三条带。其分子量分别为37,78,与120kDa。经初步计算,RGD-TRAIL-ELP三聚体占总量的38%(图1B),RGD-TRAIL三聚体含量仅为12%。因而融合ELP之后,有助于TRAIL形成更多的三聚体。
RGD-TRAIL-ELP分别在37与10℃检测其粒径分布。在10℃时,RGD-TRAIL-ELP的平均粒径只有8nm,当温度为37℃时,其粒径升至190nm(图2A),冷冻蚀刻-透射电镜观察RGD-TRAIL在37℃下,只有8nm(图2B),与10℃时,RGD-TRAIL-ELP的平均粒径接近;37℃下,RGD-TRAIL-ELP的粒径约为200nm。
在活性测试中,RGD-TRAIL作为参照。RGD-TRAIL与RGD-TRAIL-ELP半致死量(EC50)分别为1与0.35nM(20.57与13.61ng/ml),因此RGD-TRAIL-ELP诱导肿瘤细胞凋亡活性提高近3倍,这与RGD-TRAIL-ELP形成更多的三聚体有关。
评价RGD-TRAIL与RGD-TRAIL-ELP肿瘤靶向性是用COLO-205作为肿瘤模型。尾静脉注射重组蛋白24小时后,将小鼠处死后,取出肿瘤组织与其他一些重要脏器,PBS洗涤后,进行荧光定量分析。RGD-TRAIL与ELP给药组中,其肿瘤组织只检测出微弱的荧光,但在肾脏与肝脏组织中存在较强荧光信号(图4A)。而RGD-TRAIL-ELP给药组中,有强荧光在肿瘤部位富集(图4A),其荧光强度相当于2.5倍RGD-TRAIL给药组中的肿瘤组织,RGD-TRAIL-ELP给药组中的其它组织荧光较弱(图4C)
在COLO-205肿瘤模型鼠中,30μg(1.4nmol)的RGD-TRAIL低剂量给药后,同PBS相比,肿瘤生长只受到轻微抑制,而等摩尔数的RGD-TRAIL-ELP(1.4nmol,55μg)却能显著地抑制肿瘤生长,且比高剂量的RGD-TRAIL(90μg,4.2nmol)肿瘤抑制效果更明显。用ELP与RGD-TRAIL进行共注射,其产生的治疗治疗效果与单独用RGD-TRAIL给药差异不显著。尤其是当用RGD-TRAIL-ELP进行高剂量(4.3nmol,165μg)给药10天后,小鼠的肿瘤逐渐消退(图5)。
综上所述,本发明是将RGD-TRAIL与ELP融合并进行重组表达,该重组蛋白在大肠杆菌中以可溶的形式表达,该重组蛋白通过离心方法进行纯化。纯化后的重组蛋白三聚体含量高,诱导肿瘤细胞凋亡能力强,并且在生理条件下可自组装成纳米微粒,在体内产生较好的肿瘤抑制效果。本发明所述的可逆相变纯化方法简单,快速,易于规模放大,成本低廉。本发明所述的自组装方式依赖于生理条件,适用于体内环境。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种融合肿瘤坏死因子相关凋亡诱导配体TNF related apoptosis inducingligand的蛋白,即TRAIL的重组融合蛋白RGD-TRAIL-ELP,该重组融合蛋白的序列如下:
VRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGGGACDCRGDCFCGGPDGHGVGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVP。
2.根据权利要求1所述的重组融合蛋白RGD-TRAIL-ELP,所述重组融合蛋白RGD-TRAIL-ELP的特征是:由大肠杆菌表达、诱导肿瘤细胞凋亡活性高,纯化工艺简单,在生理条件下自组装成190纳米微粒,肿瘤靶向性强,抑制肿瘤生长显著。
3.根据权利要求2所述的重组融合蛋白RGD-TRAIL-ELP,所述重组融合蛋白RGD-TRAIL-ELP的特征是:所述重组融合蛋白RGD-TRAIL-ELP是肿瘤坏死因子的相关凋亡诱导配体蛋白与类弹性蛋白进行融合形成的重组蛋白;所述的肿瘤坏死因子的相关凋亡诱导配体蛋白与细胞膜上的死亡受体4与5结合诱导细胞凋亡的蛋白;所述的类弹性蛋白序列为:
GHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVP。
4.一种如权利要求1-3任意一项所述的重组融合蛋白RGD-TRAIL-ELP的可溶性表达并实现简单和快速的纯化和制备的方法,其特征为:
(1)将表达重组融合蛋白RGD-TRAIL-ELP的细菌进行蛋白表达的诱导培养,收集菌体,破碎,破碎液4度12000g离心5分钟取上清;
(2)将上清在40度下加热,40度下12000g离心取5分钟,取沉淀部分;
(3)将沉淀部分用预冷的pbs缓冲液溶解,放在4度下15分钟,4度12000g离心5分钟取上清,如此4度离心取上清及40度下取沉淀循环三次;
(4)纯化后的RGD-TRAIL-ELP经冷冻干燥之后用生理盐水溶解,蛋白质的终浓度为0.5mg/ml。
5.如权利要求4所述的一种重组融合蛋白RGD-TRAIL-ELP的可溶性表达并实现简单和快速的纯化和制备的方法,其特征为:步骤(1)所述的将表达重组融合蛋白RGD-TRAIL-ELP的细菌进行蛋白表达的诱导培养为:含有重组融合蛋白RGD-TRAIL-ELP表达质粒的大肠杆菌生长温度为37度,待长至OD为0.6时,将温度调整为30度,加入终浓度为0.5mM的IPTG诱导剂,诱导16小时。
6.如权利要求1-3任意一项所述的重组融合蛋白RGD-TRAIL-ELP的应用方法,其特征是用于腹腔注射或者静脉注射。
7.如权利要求1-3任意一项所述的重组融合蛋白RGD-TRAIL-ELP在制备抗肿瘤药物方面的应用。
8.如权利要求1-3任意一项所述的重组融合蛋白RGD-TRAIL-ELP在制备诱导肿瘤细胞凋亡方面的应用。
9.一种放大RGD-TRAIL蛋白粒径的方法,其特征为:在融合蛋白RGD-TRAIL蛋白的末端融合ELP序列,融合之后的蛋白为RGD-TRAIL-ELP,所述ELP序列为:
GHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVP;所述融合蛋白RGD-TRAIL-ELP通过自组装行为实现放大蛋白的粒径:在10℃时,RGD-TRAIL-ELP的平均粒径只有8nm,当温度为37℃时,其自组装之后的粒径升至190nm,可以由8nm放大为190nm,有效放大了RGD-TRAIL蛋白的粒径。
10.一种实现融合蛋白RGD-TRAIL可溶性表达、且简单和快速的纯化的方法,其特征为:
在融合蛋白RGD-TRAIL蛋白的末端融合ELP序列,融合之后的蛋白为RGD-TRAIL-ELP,所述ELP序列为:
GHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVP,由于所融合的ELP序列在细菌破碎之后,在40度下会自组装发生聚集变成沉淀;而在4度下15分钟又可以解聚集,成为溶解的蛋白,因此可以利用此特性实现可溶性表达、且简单和快速的纯化,具体的步骤如下:
(1)将表达重组融合蛋白RGD-TRAIL-ELP的细菌进行蛋白表达的诱导培养,收集菌体,破碎,破碎液4度12000g离心5分钟取上清;
(2)将上清在40度下加热,40度下12000g离心取5分钟,取沉淀部分;
(3)将沉淀部分用预冷的pbs缓冲液溶解,放在4度下15分钟,4度12000g离心5分钟取上清,如此4度离心取上清及40度下取沉淀循环三次;
(4)纯化后的RGD-TRAIL-ELP经冷冻干燥之后用生理盐水溶解,蛋白质的终浓度为0.5mg/ml;
其中上述步骤(1)所述的将表达重组融合蛋白RGD-TRAIL-ELP的细菌进行蛋白表达的诱导培养为:含有重组融合蛋白RGD-TRAIL-ELP表达质粒的大肠杆菌生长温度为37度,待长至OD为0.6时,将温度调整为30度,加入终浓度为0.5mM的IPTG诱导剂,诱导16小时。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
WO2019154230A1 (zh) * | 2018-02-08 | 2019-08-15 | 四川大学华西医院 | 一种包含TRAIL和IgG结合结构域的融合蛋白及其用途 |
CN110551205A (zh) * | 2018-06-01 | 2019-12-10 | 云南大学 | 可溶性人肿瘤坏死因子凋亡相关诱导配体的突变体及其制备方法和应用 |
CN114796516A (zh) * | 2022-04-20 | 2022-07-29 | 台州学院 | 一种多功能蛋白抗肿瘤纳米药物及制备方法和应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101418290A (zh) * | 2008-12-02 | 2009-04-29 | 中山大学 | 一种高效的elp融合蛋白酶及其制备和应用 |
CN101578373A (zh) * | 2006-09-06 | 2009-11-11 | 费斯生物制药公司 | 融合肽治疗组合物 |
CN102775497A (zh) * | 2012-07-13 | 2012-11-14 | 浙江大学 | 肿瘤坏死因子相关的凋亡诱导配体融合蛋白及其制备方法 |
CN104725515A (zh) * | 2015-03-20 | 2015-06-24 | 扬州大学 | 一种类弹性蛋白多肽与柯萨奇腺病毒受体融合蛋白及其制备方法和应用 |
CN105175554A (zh) * | 2015-10-26 | 2015-12-23 | 扬州大学 | 一种类弹性蛋白多肽与热应激蛋白90α融合蛋白及其制备方法和应用 |
CN105727261A (zh) * | 2008-06-27 | 2016-07-06 | 杜克大学 | 包含弹性蛋白样肽的治疗剂 |
-
2016
- 2016-11-02 CN CN201610951811.9A patent/CN106519040B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101578373A (zh) * | 2006-09-06 | 2009-11-11 | 费斯生物制药公司 | 融合肽治疗组合物 |
CN105727261A (zh) * | 2008-06-27 | 2016-07-06 | 杜克大学 | 包含弹性蛋白样肽的治疗剂 |
CN101418290A (zh) * | 2008-12-02 | 2009-04-29 | 中山大学 | 一种高效的elp融合蛋白酶及其制备和应用 |
CN102775497A (zh) * | 2012-07-13 | 2012-11-14 | 浙江大学 | 肿瘤坏死因子相关的凋亡诱导配体融合蛋白及其制备方法 |
CN104725515A (zh) * | 2015-03-20 | 2015-06-24 | 扬州大学 | 一种类弹性蛋白多肽与柯萨奇腺病毒受体融合蛋白及其制备方法和应用 |
CN105175554A (zh) * | 2015-10-26 | 2015-12-23 | 扬州大学 | 一种类弹性蛋白多肽与热应激蛋白90α融合蛋白及其制备方法和应用 |
Non-Patent Citations (5)
Title |
---|
HUA, ZICHUN等: "Fused hydrophobic elastin-like-peptides (ELP) enhance biological activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)", 《PROTEIN & PEPTIDE LETTERS》 * |
HYMOWITZ,S.G等: "Chain A, Crystal Structure Of Death Receptor 5 (Dr5) Bound To Apo2lTRAIL", 《GENBANK》 * |
葛慧华等: "影响类弹性蛋白多肽自组装成微球的因素及其作用机制", 《生物工程学报》 * |
董文凤等: "类弹性蛋白多肽在给药系统中的应用研究进展", 《药学服务与研究》 * |
黄凯宗等: "类弹性蛋白多肽的从头设计、非色谱纯化及盐效应", 《生物工程学报》 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019154230A1 (zh) * | 2018-02-08 | 2019-08-15 | 四川大学华西医院 | 一种包含TRAIL和IgG结合结构域的融合蛋白及其用途 |
US11952402B2 (en) | 2018-02-08 | 2024-04-09 | West China Hospital, Sichuan University | Fusion protein containing trail and IgG binding domain and the uses thereof |
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 |
CN108587998B (zh) * | 2018-05-17 | 2021-05-18 | 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 |
CN110551205A (zh) * | 2018-06-01 | 2019-12-10 | 云南大学 | 可溶性人肿瘤坏死因子凋亡相关诱导配体的突变体及其制备方法和应用 |
CN110551205B (zh) * | 2018-06-01 | 2023-07-28 | 云南大学 | 可溶性人肿瘤坏死因子凋亡相关诱导配体的突变体及其制备方法和应用 |
CN114796516A (zh) * | 2022-04-20 | 2022-07-29 | 台州学院 | 一种多功能蛋白抗肿瘤纳米药物及制备方法和应用 |
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