CN110256577A - 一种融合白蛋白纳米粒及其应用 - Google Patents
一种融合白蛋白纳米粒及其应用 Download PDFInfo
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- CN110256577A CN110256577A CN201910525910.4A CN201910525910A CN110256577A CN 110256577 A CN110256577 A CN 110256577A CN 201910525910 A CN201910525910 A CN 201910525910A CN 110256577 A CN110256577 A CN 110256577A
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Abstract
本发明公开了一种融合白蛋白纳米粒及其应用,属于生物医药技术领域。本发明利用基因工程技术表达融合白蛋白,并利用该融合白蛋白在中性水溶液中自组装载药,形成融合白蛋白纳米粒。本发明所研究出的融合白蛋白综合了靶向、pH和酶敏感官能团,利用此融合白蛋白制备得纳米粒具备靶向和可控释药的功能,该融合白蛋白纳米粒的制备方法简单易行,具有巨大的应用潜力。
Description
技术领域
本发明涉及一种融合白蛋白纳米粒及其应用,属于生物医药技术领域。
背景技术
白蛋白为内源性物质,并且是一种不具有调理作用的蛋白。早期的研究发现,将其包覆于纳米粒或脂质体表面,可降低微粒对巨噬细胞的亲和力,从而延长循环时间,提高靶向性。结合了纳米粒载体和白蛋白性质两方面优势应运而生的白蛋白纳米粒载药系统近年来受到广泛关注。白蛋白纳米粒是以白蛋白作为载体,包封或吸附药物,经过固化分离形成的实心球体,具有生物相容性良好、毒性及刺激性低、无抗原性等多种优点。白蛋白纳米粒能够包裹的药物有抗肿瘤药、抗结核药、降血糖药、抗菌素等,目前最引人注目的应用还是将其作为抗肿瘤药物的载体,增加靶向性,减小毒副作用,提高疗效。
由美国AbraxisBioScienceInc开发的紫杉醇人血清白蛋白纳米粒注射剂获得FDA批准上市,成为首个白蛋白纳米粒载药系统的成功案例。是将紫杉醇与人血清白蛋白结合,经静脉注射进入人体循环后,其很快分解成更小的白蛋白-紫杉醇复合物,利用天然白蛋白通路实现药物靶向并进入肿瘤细胞。主要是利用美国AmericanBioscienceInc.公开的一种独特的基于二硫键形成法的NabTM技术(nanoparticle.albuminboundtechnology,NabTM_technology)NabTM-技术是以白蛋白作为基质和稳定剂,在高剪切力(例如,超声处理、高压均化或类似方法)下,将包含水不溶性药物的油相和含白蛋白的水相混合,制备O/W乳剂,在没有任何常规表面活性剂或任何聚合物核心存在的情况下制备药物的白蛋白纳米粒的技术。
然而,面临着一些不可避免的问题:首先,NabTM技术需要使用氯仿、二氯甲烷等有毒溶剂,且存在制备方法周期长及工艺繁琐等缺点;其次,白蛋白原料的获得受血液来源及提取污染的限制,且同一制备方法中,白蛋白的批间差异、白蛋白浓度、溶剂、温度、交联剂等会影响最终纳米制剂的性质;再次,是以非晶态、无定型状态存在,经静脉注射进人体循环后,纳米粒很快分解成更小的白蛋白-紫杉醇复合物,具有提前释药的风险。另外,目前上市白蛋白纳米粒的制备工艺较为复杂,需要高压均质以获得粒径合适、均一的纳米粒。
发明内容
为解决现有技术存在的上述缺陷,制备一种可控释药的载体,本发明通过重组蛋白技术制备可控释药型融合白蛋白纳米,并提供该融合白蛋白纳米在制备靶向药物方面的应用。
本发明的第一个目的在于提供一种融合白蛋白纳米粒,所述融合白蛋白纳米粒是将RGD、白蛋白(HSA)、基质金属蛋白酶(MMP)和6~18个组氨酸依次连接,融合表达;所述RGD、白蛋白和基质金属蛋白酶通过连接肽连接。
在本发明的一种实施方式中,所述连接肽的氨基酸序列是DDDDK。
在本发明的一种实施方式中,编码所述连接肽的核苷酸序列是gatgatgatgataag。
在本发明的一种实施方式中,所述白蛋白是人血清蛋白。
在本发明的一种实施方式中,所述融合白蛋白含有SEQ ID NO.1所示的氨基酸序列。
本发明的第二个目的是提供编码所述融合白蛋白的基因。
本发明的第三个目的是提供表达所述融合白蛋白的细胞。
在本发明的一种实施方式中,所述细胞包括细菌细胞或真菌细胞。
在本发明的一种实施方式中,所述细胞为酵母细胞。
在本发明的一种实施方式中,所述酵母为毕赤酵母SMD1168H。
本发明的第四个目的是提供一种制备可控释药型融合白蛋白纳米载体的方法,所述方法是将编码所述融合白蛋白的基因与载体连接,转化至毕赤酵母细胞中。
在本发明的一种实施方式中,所述载体是pPICZαA。
在本发明的一种实施方式中,所述载体上连接信号肽α-factor。
在本发明的一种实施方式中,所述融合白蛋白纳米载体是以毕赤酵母SMD1168H为宿主,以pPICZαA为载体表达的如SEQ ID NO.1所示的融合白蛋白。
在本发明的一种实施方式中,所述方法是将表达了所述融合白蛋白的毕赤酵母发酵,收集发酵液,提取蛋白。
在本发明的一种实施方式中,所述方法还对提取的蛋白进行纯化。
本发明的第五个目的是提供所述方法制备的可控释药型融合白蛋白纳米载体在装载药物方面的应用。
在本发明的一种实施方式中,所述应用是用pH响应自组装技术装载药物。
在本发明的一种实施方式中,所述药物包括但不限于紫杉醇、多西他赛、阿霉素。
在本发明的一种实施方式中,所述应用是装载siRNA。
在本发明的一种实施方式中,所述装载是将所述白蛋白与药物以1:2的比例在pH为5~6的条件下混合,而后pH调节至7~8。
在本发明的一种实施方式中,所述装载是将所述白蛋白与药物以1:2比例在pH为5.5的条件下混合,而后pH调节至7.4。
本发明的第六个目的是提供应用所述可控释药型融合白蛋白纳米载体包载的药物。
本发明还要求保护所述融合白蛋白在制备预防或治疗肝癌,胃癌,肺癌、乳腺癌、宫颈癌等抗癌的药物中的应用。
在本发明的一种实施方式中,所述药物作用对象包括但不限于HepG2,MGC-803,A549,MCF-7,HeLa细胞。
有益效果:本发明利用基因工程技术表达一系列白蛋白融合蛋白(RGD-link-HSA-link-MMP-His),该融合白蛋白在可自组装载药形成白蛋白融合蛋白纳米粒。首先,重组蛋白技术可以使细胞本身变成“批量生产药物的工厂”,确保白蛋白来源的安全可靠;其次本发明所研究出的融合白蛋白综合了靶向、pH和酶敏感官能团,利用此融合白蛋白制备得纳米粒具备靶向和可控释药的功能,且制备融合白蛋白纳米粒的方法简单易行,适合工业化应用。
附图说明
图1(A)各融合基因序列及命名;(B)构建的pPICZαA-RGD-HSA-MMP-His载体,目的基因插入在Xho I和Not I之间;(C)融合PCR后扩增获得的各基因序列的核酸凝胶电泳图;(D)各组白蛋白融合蛋白的Western Blot分析,从上到下分别为HSA,His,RGD特异性分析;(E)RHMH18的基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分子量测定结果。
图2为各载药白蛋白融合蛋白纳米粒子透射电镜图。
图3为各载药白蛋白融合蛋白纳米粒子对代表性细胞的毒性及细胞对粒子的摄取情况:(A)各载药白蛋白融合蛋白纳米粒子对正常细胞293的毒性测试结果;(B)各载药白蛋白融合蛋白纳米粒子对癌细胞HepG2的毒性测试结果;(C)各载药白蛋白融合蛋白纳米粒子对癌细胞MGC-803的毒性测试结果;(D)癌细胞HepG2对最终纳米粒子(RHMH18)的摄取情况。
具体实施方式
毕赤酵母(Pichia pastoris)表达菌株SMD1168H、大肠杆菌菌株DH5α为本实验室保存。
YPD液体(发酵)培养基:1%(w/w)酵母粉,2%(w/w)蛋白胨,2%(w/w)葡萄糖。
YPD固体(平板)培养基:1%(w/w)酵母粉,2%(w/w)蛋白胨,2%(w/w)葡萄糖,1%(w/w)琼脂。
含有机碳源甘油的培养基BMGY:1%(w/w)酵母粉,2%(w/w)蛋白胨,1%(v/v)甘油,KPB缓冲液(磷酸钾盐)100mmol,10%(v/v)YNB10×;
含无机碳源甲醇的培养基BMMY:1%(w/w)酵母粉,2%(w/w)蛋白胨,0.5%(v/v)甲醇,KPB缓冲液(磷酸钾盐)100mmol,10%(v/v)YNB 10×;
LB液体培养基:1%(w/w)蛋白胨,0.5%(w/w)酵母粉,0.5%(w/w)氯化钠;
LB固体培养基:1%(w/w)蛋白胨,0.5%(w/w)酵母粉,0.5%(w/w)氯化钠,1.5%(w/w)琼脂粉。
实施例1pPICZαA重组质粒的构建
以酿酒酵母的α分泌信号肽(α-linker)为引导肽,如图1A设计的pPICZαA/Doublelinker(氨基酸序列为gatgatgatgataag)在XhoI和NotI酶切位点中加入所需的表达框架。
以pPICZαA-HSA(核苷酸序列如SEQ ID NO.14所示)为模板,以表1的引物PCR扩增目的基因。经过PCR扩增分别获得3RGD-link-HSA-link-MMP-18His,3RGD-link-HSA-link-MMP-6His,3RGD-HSA,3RGD-link-HSA-link-18His,HSA-link-MMP-18His,HSA-18His的基因片段,PCR所用的引物见表1,下划线的基因序列为酶切位点序列。反应条件见表2。
表1目标基因扩增引物表
表2 PCR扩增体系及条件
将获得的一系列基因片段分别通过核酸凝胶电泳分离出目标片段,接着利用柱式DNA胶试剂盒回收,将回收到的DNA片段于37℃金属浴双酶切30min后,利用柱式PCR产物纯化试剂盒进行纯化。同时,将实验室保存的pPICZαA质粒于37℃金属浴双酶切2h,通过核酸凝胶电泳得到质粒片段,经柱式DNA胶回收试剂盒进行回收。酶切体系如表3所示。
表3目的基因和载体的酶切体系
将所得的质粒片段与目的基因片段于22℃金属浴连接1h,连接体系如表4所示。
表4酶切后的目的基因与载体的连接体系
将连接好的带有目的基因的质粒转化到DH5α中,在无抗性的LB培养基中(500μL),37℃,200rpm摇床速度下培养1h,取100μL涂布于浓度为25μg/mL的Zeocin抗性的LB固体培养基上,培养过夜后挑选单菌落,筛选获得的阳性质粒经双酶切验证,PCR验证和测序验证。如图1B所示,构建的重组表达质粒为期望的质粒。将重组质粒送样至第三方测序公司进行测序,利用专业软件进行序列比对,结果表明,成功测序结果与预期基因序列完全一致,表明重组质粒pPICZαA-3RGD-link-HSA-link-MMP-18His构建成功。
重组质粒pPICZαA-3RGD-link-HSA-link-MMP-6His、pPICZαA-3RGD-HSA、pPICZαA3RGD-link-HSA-link-18His、pPICZαA-HSA-link-MMP-18His和pPICZαA-HSA-18His按照相同策略构建。
实施例2重组菌株的构建与筛选
将实施例1构建的重组质粒pPICZαA-18His-HSA-MMP-RGD经Sal I线性化酶切后,与SMD1168H感受态细胞混合电击(1.5kV,40μF,160Ω)转化。毕赤酵母SMD1168的感受态细胞经标准方法制备获得。在YPD平板培养四天。挑取阳性克隆,并经菌落PCR验证。分别获得表达RHMH18,RHMH6,RHH18,RH,HMH18,HH18蛋白的重组菌株。各挑选10株菌落明显的阳性克隆株到YPD液体培养基中培养三天。收集上清液用尿微量白蛋白检测试剂盒检测上清液中融合蛋白(RHMH18,RHMH6,RHH18,RH,HMH18,HH18)的蛋白含量,并挑选高表达的克隆菌株。
筛选出来的菌株的发酵上清经WB验证后,结果显示同时具有His、RGD和HSA的抗原性,发酵上清的蛋白为Double linker的融合蛋白,且分子大小为71796Da,与预计的一致(图1E)。提取酵母的基因组,PCR扩增结果显示,目标基因存在于酵母基因组中。结果表明筛选获得的菌株为表达目标蛋白的菌株,后续的研究以此筛选菌株作为出发重组菌株。
实施例3重组蛋白的制备与纯化
筛选获得的菌株接种至小摇瓶中在28℃、200rpm培养1d,制备种子液。在装液量为300mL BMGY的1L的三角摇瓶中接种,接种量为2%(v/v)。在28℃、200rpm培养1d,静置过夜后将发酵上清置换为BMMY诱导蛋白分泌。每天补加1%(v/v)的甲醇,连续在28℃、150rpm培养4d后,8000rpm离心10min发酵上清即为我们需要的目标蛋白。
实施例4重组蛋白的表达和纯化
发酵液经8000rpm离心10min,离心收集发酵上清,并用0.45μm的滤膜过滤除菌。再用截留分子量为10kDa的超滤仪超滤浓缩10倍。补加两次等体积的水,再次浓缩。BlueSepharose先经A液(20mM NaPB pH 7.2,0.1M NaCl)平衡。离心获得的发酵上清经0.45μm的滤膜过滤后,将发酵上清上样至Blue柱,用A液淋洗平衡后先用100%B液(20mM NaPB pH7.2,2M NaCl)洗脱,再用C液(1M Arg,pH 7.2)强洗。G25脱盐柱将缓冲液置换成D液(25mMTris-HCl,50mM NaCl,2mM CaCl2,pH 7.6),将溶液加入3000分子量的透析袋中,透析3天后,在-20℃预冻后放入冻干机冻干。
实施例5重组蛋白的功能验证
制备好12%的SDS-PAGE胶,离心收集发酵上清液。处理样品后上样,电泳条件为150V、80min,并转化到硝酸纤维素膜上。制备三份,Marker范围为10kDa-180kDa,并分别用His单抗、RGD多抗和HSA多抗杂交,用羊抗小鼠的二抗孵育。孵育好的硝酸纤维素膜用ECL(chemiluminescent reagent)显色液显色。结果显示,所有融合蛋白均出现了HSA的特异性条带,融合有RGD或His的样品分别出现相应的特异性条带,表明白蛋白融合蛋白成功表达了相应的功能组分(RGD,His)。
实施例6重组纳米粒子的形貌分析
利用透射电镜观察重组融合蛋白粒子的形态结构:取一定量各组重组融合蛋白粒子加适量超纯水溶液超声充分分散,用移液枪吸取混悬液滴于覆有碳膜的铜筛网上,自然晾干后置于透射电镜下观察。
如图2所示,形成的重组蛋白纳米粒子呈圆形,直径为100nm,其中RHMH18形成的粒子可看到清晰的外形轮廓,粒子形状均一规则,其余粒子的外形轮廓的光滑程度不及RHMH18。
实施例7重组融合蛋白包载紫杉醇
经冷冻干燥机冻干后的白蛋白融合蛋白粉末溶于PBS缓冲溶液中配置成1mg/mL的溶液,用1mM的HCl溶液调节pH值至5.5,加入10mg/mL的紫杉醇溶液,用1mM的NaOH溶液调节pH值至7.4,10000rpm离心20min,得到的沉淀用超纯水混匀,1000rpm离心20min,重复上述步骤3次,即得到载紫杉醇融合蛋白纳米粒子。RHMH18,RHMH6,RHH18,HMH18,HH18,RH的药物包载率为6.59%,3.21%,5.88%,5.64%,5.09%,6.38%。
实施例8重组融合蛋白包载多西他赛
经冷冻干燥机冻干后的白蛋白融合蛋白粉末溶于PBS缓冲溶液中配置成1mg/mL的溶液,用1mM的HCl溶液调节pH值至5.5,加入10mg/mL的多西他赛溶液,用1mM的NaOH溶液调节pH值至7.4,10000rpm离心20min,得到的沉淀用超纯水混匀,1000rpm离心20min,重复上述步骤3次,即得到融合蛋白纳米粒子。RHMH18,RHMH6,RHH18,HMH18,HH18,RH的药物包载率为7.31%,4.87%,7.06%,6.34%,6.17%,7.08%。
实施例9重组融合蛋白的药物的体外释放
分别称取适量各纳米粒子溶于对应PBS缓冲液中.将混合体系转移到透析袋(3500KD)中,用棉绳扎紧口部.透析袋放置在含10倍体积PBS的EP管里,在37℃、100rpm恒温摇床里振荡,分别于10min、20min、30min、1h、2h、3h、5h、7h、9h、11h、24h、27h、48h吸取3mL释放液,同时补加同温度、同pH的缓冲液3mL。收集标记各个时间点的释放液,用紫外分光光度计测量吸收值.根据标准曲线计算PTX的累计释放率,以累计释放率对时间绘图可得阿霉素体外释放曲线。
实施例10重组融合蛋白的药物毒性实验
以包载紫杉醇的白蛋白融合蛋白纳米粒子为例:将HEK 293,HepG2和MGC-803三种细胞在含有10%胎牛血清和1%双抗的DMEM培养基中胞置于在37℃,5%CO2的培养箱中培养,2天传代1次,取对数生长期的细胞进行实验。
取对数生长期的细胞进行消化并计数,以5×103/孔的密度接种于96孔板中进行培养。细胞贴壁后,将完全培养基换成100μL含有药物浓度分别为10μg/mL,20μg/mL,30μg/mL,40μg/mL和50μg/mL的重组蛋白溶液,孵育24h后将溶液吸出,用PBS小心润洗三次,除去未被细胞内吞的材料,再加入100μL 0.5mg/mL MTT溶液,置于培养箱继续孵育4h,吸去MTT溶液,加入DMSO并振荡15min,置于酶标仪上检测。以未加纳米粒子的空白组作为对照。每个实验重复三次,取平均值。结果显示,相比较游离紫杉醇及没有融合His的蛋白纳米粒子(Abraxane,RH),其他融合蛋白样品对正常细胞显示出较好的生物相容性,细胞存活率均在70%以上;针对HepG2癌细胞,相比较市售Abraxane,载紫杉醇RHMH18呈现更强的毒性,粒子浓度为50μg/mL时细胞存活率为30%左右;针对MGC-803癌细胞,载紫杉醇RHMH18呈现与Abraxane相当的细胞毒性,粒子浓度为50μg/mL时细胞存活率为20%左右。
实施例11重组融合蛋白细胞摄取
以包载紫杉醇的白蛋白融合蛋白纳米粒子为例:将对数生长期的HEK 293,HepG2,MGC-803细胞分别以3×105个/孔的密度接种于6孔板中。细胞贴壁后,将培养基换成修饰有罗丹明B的重组蛋白的培养基溶液(50μg/mL),孵育2h后将溶液吸出,用PBS小心润洗三次,除去未被细胞内吞的材料,再将细胞用胰酶消化,重悬于PBS中,在流式细胞仪中进行定量测试。结果显示,相比较其他融合蛋白纳米粒,三种细胞均对RHMH18呈现更强的摄取。
另外,细胞贴壁后,将培养基换成含有药物浓度分别为10μg/mL,20μg/mL,30μg/mL,40μg/mL和50μg/mL的重组蛋白的培养基溶液,孵育不同时间后将溶液吸出,用PBS小心润洗,用胰酶消化后PBS重悬,置于流式细胞仪中测试。以未加蛋白粒子的空白组作为对照进行调试。凋亡结果显示,相比较其他融合蛋白纳米粒,载紫杉醇RHMH18对癌细胞呈现更强的杀伤能力。
将HEK 293,HepG2,MGC-803以2×105个/孔的密度接种于共聚焦用细胞培养皿中,加入含有重组蛋白培养基溶液(400mg/L),共孵育一定时间后,用PBS润洗三次,之后加入多聚甲醛溶液(4%,共孵育20min固定细胞,再加入DAPI溶液(200ng/mL,置于培养箱30min对细胞核进行染色,最后用PBS润洗三次,用CLSM进行定性观测。以HepG2为代表细胞进行分析,结果显示随着培养时间的推移,RHMH18跨过细胞膜,逐渐分布在细胞质中,培养16小时后,粒子已经进入细胞核发挥杀伤作用。
对比例1:
白蛋白与药物(例如紫杉醇)的混合比例直接影响最终的载药率:经冷冻干燥机冻干后的融合白蛋白粉末溶于PBS缓冲溶液中配置成1mg/mL的溶液,用1mM的HCl溶液调节pH值至5.5,分别加入5mg/mL,8mg/mL,12mg/mL,15mg/mL的药物溶液,用1mM的NaOH溶液调节pH值至7.4,10000rpm离心20min,得到的沉淀用超纯水混匀,1000rpm离心20min,重复上述步骤3次,即得到载紫杉醇融合蛋白纳米粒子。经测试,相比较于药物浓度为10mg/mL,当药物浓度为5mg/mL和8mg/mL时,纳米粒子的载药效率大幅度下降;当药物浓度为12mg/mL和15mg/mL,纳米粒子的载药效率相当。
对比例2:
白蛋白与药物(例如紫杉醇)的混合时pH的调节直接影响最终的载药率:经冷冻干燥机冻干后的融合白蛋白粉末溶于PBS缓冲溶液中配置成1mg/mL的溶液,用1mM的HCl溶液分别调节pH值至7,6.5,6,5,加入10mg/mL的药物溶液,用1mM的NaOH溶液调节pH值至7.4,10000rpm离心20min,得到的沉淀用超纯水混匀,1000rpm离心20min,重复上述步骤3次,即得到载紫杉醇融合蛋白纳米粒子。经测试,RHMH18的载药量分别为2.35%,4.14%,5.21%,5.19%。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种融合白蛋白纳米粒及其应用
<160> 14
<170> PatentIn version 3.3
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<213> 人工序列
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Leu Glu Lys Arg Arg Gly Asp Arg Gly Asp Arg Gly Asp Asp Asp Asp
1 5 10 15
Asp Lys Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu
20 25 30
Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr
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Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
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Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys
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Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala
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Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln
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Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
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Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala
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Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile
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Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala
165 170 175
Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys
180 185 190
Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
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Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
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Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
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Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
245 250 255
Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala
260 265 270
Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser
275 280 285
Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
290 295 300
Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu
305 310 315 320
Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
325 330 335
Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr
340 345 350
Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
355 360 365
Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro
370 375 380
His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu
385 390 395 400
Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu
405 410 415
Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys
420 425 430
Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu
435 440 445
Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
450 455 460
Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val
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Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
485 490 495
Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp
500 505 510
Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
515 520 525
Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
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Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu
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Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
565 570 575
Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys
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Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Asp Asp Asp Asp Lys
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Pro Leu Gly Leu Trp Ala His His His His His His His His His His
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His His His His His His His His Ala Ala
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ttgcggccgc ttaatggtga tggtgatggt gatggtgatg gtgatggtga tggtgatggt 60
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ttgcagcaac 130
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ccctcgagaa aagacgcgga gatcgcggag atcgcggaga tgatgatgat gataaggatg 60
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ttgcggccgc ttaatggtga tggtgatggt gtgcccataa tcctaatggc ttatcatcat 60
catctaagcc taaggcagct tgacttgcag caac 94
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<212> DNA
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ccctcgagaa aagacgcgga gatcgcggag atcgcggaga tgatgcacac aagagtgagg 60
ttgctcatcg atttaaagat ttgggagaag 90
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ttgcggccgc ttaatggtga tggtgatggt gatggtgatg gtgatggtga tggtgatggt 60
gatggtgtgc ccataatcct aatggtaagc ctaaggcagc ttgacttgca gcaacaagtt 120
ttttac 126
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ccctcgagaa aagacgcgga gatcgcggag atcgcggaga tgatgatgat gataaggatg 60
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ttgcggccgc ttaatggtga tggtgatggt gatggtgatg gtgatggtga tggtgatggt 60
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<210> 10
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ccctcgagaa aagagatgca cacaagagtg aggttgctca tcga 44
<210> 11
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<213> 人工序列
<400> 11
ttgcggccgc ttaatggtga tggtgatggt gatggtgatg gtgatggtga tggtgatggt 60
gatggtgtgc ccataatcct aatggcttat catcatcatc taagcctaag gcagcttgac 120
ttgcagcaac 130
<210> 12
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<210> 13
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ttgcggccgc ttaatggtga tggtgatggt gatggtgatg gtgatggtga tggtgatggt 60
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agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt 960
tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga 1020
agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga 1080
tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa 1140
tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagacgcgg 1200
agatcgcgga gatcgcggag atgatgatga tgataaggat gcacacaaga gtgaggttgc 1260
tcatcgattt aaagatttgg gagaagaaaa tttcaaagcc ttggtgttga ttgcctttgc 1320
tcagtatctt cagcagtgtc catttgaaga tcatgtaaaa ttagtgaatg aagtaactga 1380
atttgcaaaa acatgtgttg ctgatgagtc agctgaaaat tgtgacaaat cacttcatac 1440
cctttttgga gacaaattat gcacagttgc aactcttcgt gaaacctatg gtgaaatggc 1500
tgactgctgt gcaaaacaag aacctgagag aaatgaatgc ttcttgcaac acaaagatga 1560
caacccaaac ctcccccgat tggtgagacc agaggttgat gtgatgtgca ctgcttttca 1620
tgacaatgaa gagacatttt tgaaaaaata cttatatgaa attgccagaa gacatcctta 1680
cttttatgcc ccggaactcc ttttctttgc taaaaggtat aaagctgctt ttacagaatg 1740
ttgccaagct gctgataaag ctgcctgcct gttgccaaag ctcgatgaac ttcgggatga 1800
agggaaggct tcgtctgcca aacagagact caagtgtgcc agtctccaaa aatttggaga 1860
aagagctttc aaagcatggg cagtagctcg cctgagccag agatttccca aagctgagtt 1920
tgcagaagtt tccaagttag tgacagatct taccaaagtc cacacggaat gctgccatgg 1980
agatctgctt gaatgtgctg atgacagggc ggaccttgcc aagtatatct gtgaaaatca 2040
agattcgatc tccagtaaac tgaaggaatg ctgtgaaaaa cctctgttgg aaaaatccca 2100
ctgcattgcc gaagtggaaa atgatgagat gcctgctgac ttgccttcat tagctgctga 2160
ttttgttgaa agtaaggatg tttgcaaaaa ctatgctgag gcaaaggatg tcttcctggg 2220
catgtttttg tatgaatatg caagaaggca tcctgattac tctgtcgtgc tgctgctgag 2280
acttgccaag acatatgaaa ccactctaga gaagtgctgt gccgctgcag atcctcatga 2340
atgctatgcc aaagtgttcg atgaatttaa acctcttgtg gaagagcctc agaatttaat 2400
caaacaaaat tgtgagcttt ttgagcagct tggagagtac aaattccaga atgcgctatt 2460
agttcgttac accaagaaag taccccaagt gtcaactcca actcttgtag aggtctcaag 2520
aaacctagga aaagtgggca gcaaatgttg taaacatcct gaagcaaaaa gaatgccctg 2580
tgcagaagac tatctatccg tggtcctgaa ccagttatgt gtgttgcatg agaaaacgcc 2640
agtaagtgac agagtcacca aatgctgcac agaatccttg gtgaacaggc gaccatgctt 2700
ttcagctctg gaagtcgatg aaacatacgt tcccaaagag tttaatgctg aaacattcac 2760
cttccatgca gatatatgca cactttctga gaaggagaga caaatcaaga aacaaactgc 2820
acttgttgag ctcgtgaaac acaagcccaa ggcaacaaaa gagcaactga aagctgttat 2880
ggatgatttc gcagcttttg tagagaagtg ctgcaaggct gacgataagg agacctgctt 2940
tgccgaggag ggtaaaaaac ttgttgctgc aagtcaagct gccttaggct tagatgatga 3000
tgataagcca ttaggattat gggcacacca tcaccatcac catcaccatc accatcacca 3060
tcaccatcac catcaccatt aagcggccgc cagctttcta gaacaaaaac tcatctcaga 3120
agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt gtagccttag 3180
acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt gctagattct 3240
aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt 3300
ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga 3360
gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa 3420
aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat 3480
taagtgagac cttcgtttgt gcggatcccc cacacaccat agcttcaaaa tgtttctact 3540
ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac 3600
ccaagcacag catactaaat tttccctctt tcttcctcta gggtgtcgtt aattacccgt 3660
actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg 3720
caataaaaat ttttatcacg tttctttttc ttgaaatttt tttttttagt ttttttctct 3780
ttcagtgacc tccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt 3840
ttcatttttc ttgttctatt acaacttttt ttacttcttg ttcattagaa agaaagcata 3900
gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt 3960
ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct 4020
caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg 4080
ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag 4140
cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct 4200
ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg 4260
gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc 4320
ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacggcggcc 4380
cacgggtccc aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag 4440
ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag 4500
ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa 4560
cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca 4620
ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc 4680
tggagaccaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 4740
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 4800
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 4860
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 4920
cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 4980
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 5040
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 5100
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 5160
agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 5220
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 5280
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 5340
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 5400
ggattttggt catgagatc 5419
Claims (10)
1.一种融合蛋白,其特征在于,依次含有RGD、白蛋白、基质金属蛋白酶和6~18个组氨酸;所述RGD、白蛋白和基质金属蛋白酶通过连接肽连接。
2.根据权利要求1所述的融合蛋白,其特征在于,所述连接肽的氨基酸序列含有DDDDK;所述白蛋白是人血清蛋白。
3.编码权利要求1或2所述融合蛋白的基因。
4.表达权利要求1或2所述融合蛋白的细胞。
5.一种制备可控释药型融合白蛋白纳米载体的方法,其特征在于,所述方法是将编码所述融合白蛋白的基因与载体连接,转化至毕赤酵母细胞中。
6.一种药物组合物,其特征在于,应用权利要求1或2所述的融合白蛋白装载药物。
7.根据权利要求6所述的药物组合物,其特征在于,采用pH响应自组装技术装载药物或siRNA;所述药物包括但不限于紫杉醇、多西他赛、阿霉素。
8.制备权利要求6或7所述药物组合物的方法,其特征在于,将融合白蛋白与药物以1:2-10比例在pH为5~6的条件下混合,而后pH调节至7~8。
9.权利要求1或2所述的融合白蛋白在制备预防或治疗包括肝癌,胃癌,肺癌、乳腺癌或宫颈癌在内的药物中的应用。
10.权利要求1或2所述的融合白蛋白在制备肿瘤细胞抑制剂方面的应用,其特征在于,所述肿瘤细胞包括但不限于HepG2、MGC-803、A549、MCF-7或HeLa细胞。
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