CN116410333A - 一类促凋亡铁蛋白纳米粒和应用 - Google Patents
一类促凋亡铁蛋白纳米粒和应用 Download PDFInfo
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- CN116410333A CN116410333A CN202111682881.6A CN202111682881A CN116410333A CN 116410333 A CN116410333 A CN 116410333A CN 202111682881 A CN202111682881 A CN 202111682881A CN 116410333 A CN116410333 A CN 116410333A
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Abstract
本发明提供了一种促凋亡铁蛋白纳米粒及其应用,属于基因工程与蛋白质工程领域。本发明以促凋亡蛋白质BAK的BH3序列和RGD肽、人铁蛋白重链亚基进行重组表达,构建具有靶向和跨越血脑屏障、促凋亡的蛋白质自组装纳米粒。该纳米粒对大鼠胶质瘤细胞(C6细胞)和小鼠脑微血管内皮细胞(bEnd.3细胞)具有抑制作用,具有潜在的药用价值。
Description
技术领域
本发明属于基因工程与酶工程领域,具体而言,涉及一类促凋亡铁蛋白纳米粒及其应用。
背景技术
目前,全球约20%的人在其一生中会罹患恶性肿瘤,恶性肿瘤导致30-69岁的成年人死亡的比例占非传染性疾致死数的30%,研究预计到2040年将有3620万人患有恶性肿瘤,1920万人死于恶性肿瘤。因此,抗肿瘤药物的研究的需求迫切,研究进程也快速发展。目前治疗肿瘤的方式主要有手术治疗、放射治疗、化学药物治疗、免疫治疗、基因治疗等治疗方法;其中,最主要的治疗方法还是手术治疗与放疗、化疗相结合。
在肿瘤的诊断与治疗过程中,提高药物对肿瘤细胞的靶向性具有提高药物疗效、降低毒副反应的重要意义。通过肿瘤细胞表面表达的特殊受体,可以实现肿瘤药物的靶向递送。转铁蛋白受体1(TfR1)在许多肿瘤细胞过表达,例如脑胶质瘤细胞、人大肠癌癌细胞、人乳腺癌细胞、人宫颈腺癌细胞等肿瘤细胞,因此,有望通过铁蛋白与肿瘤细胞细胞膜上的TfR1相互作用,实现对脑胶质瘤等肿瘤的靶向治疗。
铁蛋白是一个分子量为450kDa的大蛋白,由24个单体自组装成内外尺寸分别为8nm和12nm的球形笼状结构,真核生物的单体分为铁蛋白重链(FHC)和铁蛋白轻链(FLC)。FHC和FLC具有高度同源性,可以形成任意比例的铁蛋白球。铁蛋白在生理环境中具有结构刚性,在高温以及一般变性剂条件下其结构都不会遭到破坏,但当pH值变为pH 2-3或极碱性pH10-12时,蛋白笼会发生可逆分解为单体,当pH值恢复到中性时,铁蛋白单体又能够重新以24个单体为组自组装成为纳米粒。基于铁蛋白纳米粒的良好生物相容性和自组装纳米粒特性,可以作为多肽或蛋白质药物的载体,提高多肽或蛋白药物的稳定性和靶向性。
发明内容
本发明要解决的技术问题是,通过蛋白质工程改造,获得具有促凋亡活性的铁蛋白自组装纳米粒。本发明通过将具有整合素αvβ3靶向性的RGD肽、促凋亡活性的BH3肽和铁蛋白HFn通过连接子(linker)构建促凋亡铁蛋白纳米粒,通过凋亡途径发挥抗肿瘤作用。
本发明具体技术方案如下:
一种重组促凋亡铁蛋白纳米粒,由具有整合素αvβ3靶向性的RGD肽、促凋亡活性的BH3肽和铁蛋白HFn通过连接子(linker)组成。
优选的,所述的重组促凋亡铁蛋白纳米粒重组蛋白RGD-BH3-HFn的氨基酸序列如SEQ ID NO.3所示。
为实现上述发明目的,根据本发明的一个方面,提供了促凋亡铁蛋白纳米粒的序列类似蛋白质BH3-HFn,SEQ ID NO.5。
根据本申请的第二个方面,提供了一类DNA分子,编码上述重组促凋亡铁蛋白纳米粒。
根据本申请的第三个方面,提供了一类重组质粒,该重组质粒连接有上述DNA分子。
根据本申请的第二个方面,提供了一类非植物的宿主细胞,该宿主细胞含有前述的重组质粒。
进一步地,宿主细胞为原核细胞,且宿主细胞为感受态细胞。
进一步地,感受态细胞为大肠杆菌BL21细胞。
本发明分离纯化的重组促凋亡铁蛋白可自组装成26.08nm左右的颗粒,具有良好的粒径分布范围。
本发明提供的促凋亡铁蛋白自组装纳米粒具有对大鼠胶质瘤细胞(C6细胞)和小鼠脑微血管内皮细胞(bEnd.3细胞)的抑制作用。
附图说明
图1为重组蛋白RGD-BH3-HFn纯化过程与SDS-PAGE电泳结果。A.阳离子纯化SDS-PAGE结果:1泳道为上株前样品;2泳道为流穿样品;3泳道为10%Buffer洗脱峰样品;4泳道为25%Buffer b洗脱峰(主峰)样品;5泳道为100%Buffer b洗脱峰样品;M,Marker。B.盐析、凝胶过滤纯化SDS-PAGE结果:1泳道为盐析复溶上清样品;4泳道为凝胶过滤上样前样品;6泳道为凝胶过滤主峰样品;M,Marker。
图2为重组蛋白RGD-BH3-HFn的纯度测定结果。
图3为重组促凋亡铁蛋白纳米粒RGD-BH3-HFn透射电子显微镜(TEM)检测结果。
图4为RGD-BH3-HFn和BH3-HFn抗肿瘤细胞活性结果。
图5为RGD-BH3-HFn和BH3-HFn促进C6细胞凋亡的流式细胞仪检测结果。
图6为RGD-BH3-HFn和BH3-HFn促进bEnd.3细胞凋亡的流式细胞仪检测结果。
具体实施方式
本发明公开了一种促凋亡铁蛋白纳米粒的氨基酸序列、制备方法、编码该蛋白的DNA分子、载体、宿主细胞,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。需要特别指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。下面结合附图和具体实施例对本发明的技术方案做进一步详细的说明,但本实施例仅用于解释本发明,而非对本发明范围的限制。
菌株和载体
大肠杆菌DH5α株、大肠杆菌BL21株、载体为pET-22b(+)。
培养基配方:大肠杆菌培养基(LB培养基):1%胰蛋白胨、0.5%酵母提取物、1%NaCl。
大肠杆菌感受态细胞转化方法。
将一步克隆产物(重组质粒)10μl加入至100μl感受态细胞中,于冰上放置30min,然后42℃热激45s,于冰上放置2min,加入0.8ml无抗生素的LB培养基,然后37℃培养1h,3000g离心3min,去掉0.6ml上清,吹打细胞沉淀将其均匀涂布在含有100μg/ml的LB固体平板上,37℃培养12-14h。
序列表编号说明:
SEQ ID NO.1为靶向整合素αvβ3的RGD肽的氨基酸序列。
SEQ ID NO.2为促凋亡活性的BH3肽的氨基酸序列。
SEQ ID NO.3为重组蛋白RGD-BH3-HFn的氨基酸序列。
SEQ ID NO.4为重组蛋白RGD-BH3-HFn的核苷酸序列。
SEQ ID NO.5为重组蛋白BH3-HFn的氨基酸序列。
SEQ ID NO.6为重组蛋白BH3-HFn的核苷酸序列。
实施例1:重组蛋白RGD-BH3-HFn的表达与纯化
将重组蛋白RGD-BH3-HFn的表达质粒转化入BL 21(DE3)pLyss感受态细胞,接种于已加入50μg/mL卡那霉素的LB固体培养基中,37℃培养24h。待菌落长至合适大小时,挑选生长良好的单菌落,接种于含有相应抗性的LB液体培养基中,放入摇床,37℃,200r/min的条件下,培养13-14h过夜活化。保存菌种后,按照6%的比例加入LB液体培养基扩大培养,37℃,200r/min培养2-3h,直至OD600达到0.7-1.0后开始诱导,加入终浓度为0.5mmol/L的IPTG,200r/min培养4h。诱导结束后9000r/min,离心5min收集菌体沉淀,菌体沉淀使用超纯水洗涤,取500μl菌样加入100μl 5x loading buffer沸水煮5min后放于-20℃冰箱备用,剩余菌样9000r/min离心10min收集菌体沉淀,放于-20℃备用。
使用高压均质机破碎菌体沉淀,提取目的蛋白:向菌体沉淀中加入适量破菌液,使用移液器重悬菌体沉淀使得沉淀与破菌液混合均匀。使用超声破碎机400w,4min进行预破碎,放入4℃冰盒保存预破碎样品。将高压均质机预冷至4℃,使用75%乙醇清洗仪器一次后,超纯水清洗仪器两、三次直至仪器内无剩余乙醇后,倒入预破碎后的菌液。800-900Bar的条件下进行高压破碎菌体沉淀,循环破碎三次直至流出较澄清的破菌液时停止破碎。收集匀浆,9000r/min,4℃离心45-60min,收集上清放于4℃待用,沉淀使用超纯水重悬后取样待用。
对破碎上清中的BH3-HFn-03蛋白进行三级纯化后冻存,具体步骤如下。
盐析纯化。将破碎所得的上清分装至50ml离心管中,分别加入上清体积1/4的4M(NH3)2SO4,4℃静置10min,目测上清逐渐浑浊,即目的蛋白析出,9000r/min,4℃离心20min,倒弃上清,收集目的蛋白沉淀,加入适量复溶盐溶液对目的蛋白进行复溶,使用移液器轻柔重悬直至目的蛋白完全复溶。使用pH计调节pH至5.00,9000r/min,4℃离心20min,收集上清使用0.22μM滤膜过滤后放于4℃冰盒待用。
使用pure 25以及UniGel-80SP对目的蛋白进行二级纯化。首先使用20%乙醇和超纯水依次清洗/>系统后,安装80SP阳离子层析柱,使用低流速检查管路通道以及层析柱是否漏液,检查无误后调节至合适流速使用超纯水清洗层析柱,待紫外和电导平稳并趋近于0后,使用Buffer b(阳离子层析Buffer 2)进行清洗,待电导上升、紫外下降并平稳后,更换为Buffer a(阳离子层析Buffer 1)平衡阳离子交换柱。待电导下降并保持平稳后开始上样,调整流速为1.5ml/min,使用a泵上样。上样结束后首先使用Buffer a冲洗至紫外下降并平稳后进行梯度洗脱,使用10%Buffer b进行杂质洗脱,25%Buffer b进行目的蛋白洗脱,并收集至50ml离心管内放于4℃冰箱待用。最后使用100%Buffer b对层析柱进行淋洗,待紫外平稳并趋近于0时,使用超纯水清洗层析柱,待电导下降并平稳后使用20%乙醇完全替换层析柱内溶液,停止运行,拆卸层析柱,使用20%乙醇清洗系统。
使用pure 25以及Superdex 75prep grade 26/600对目的蛋白进行三级纯化。首先使用20%乙醇和超纯水依次清洗/>系统后,安装Superdex 75凝胶过滤柱,使用低流速检查管路通道以及凝胶柱是否漏液,检查无误后调节至合适流速使用超纯水清洗凝胶柱,清洗100-200ml体积后使用凝胶过滤Buffer进行平衡,冲洗至电导上升并稳定后结束平衡。切换流路后使用超纯水和凝胶过滤Buffer依次清洗上样桶,待电导上升并稳定后结束清洗。使用10ml无菌一次性注射器向上样桶内注射经过一级、二级纯化的目的蛋白样品,将全部样品加入上样桶后,使用上样桶进行上样。上样结束切换系统流路使用凝胶过滤Buffer进行洗脱,待洗脱至100ml左右紫外开始上涨,待UV>50时,收集洗脱出的目的蛋白溶液。待紫外下降至50左右时,停止收集。继续使用凝胶过滤Buffer冲洗凝胶柱至电导上升并出现盐峰,电导下降后使用100-200ml超纯水清洗凝胶柱后,使用20%乙醇清洗凝胶柱至完全替换柱内溶液,停止运行,拆卸凝胶柱,使用20%乙醇清洗系统。
使用10K超滤管(Ultra-4 Centrifugal Filter Devices,Millipore)将目的蛋白浓缩至2mg/ml左右,加入9%山梨醇冻存保护剂,在超净台内使用0.22μm无菌滤膜对纯化后的目的蛋白进行过滤除菌,并用BCA法测定蛋白质浓度,标明名称日期后分装至无菌EP管内,使用液氮速冻后放于-80℃冰箱保存。纯化结果如图1所示。
实施例2:重组蛋白RGD-BH3-HFn的纯度测定
使用HPLC法对经过纯化的重组蛋白RGD-BH3-HFn进行纯度的测定,具体方法如下。
将重组蛋白RGD-BH3-HFn使用166mmol/L碳酸氢钠溶液稀释至0.5mg/ml,12000r/min离心30min后使用移液器吸取200μl上清至上样管中,注意避免加样时产生气泡。
使用100%Buffer B(乙腈,0.1%三氟乙酸)以1ml/min的流速清洗系统30min。
将流速降低至0.5ml/min,连接CAPCELL PAK Proteonavi C4柱,清洗30min。
以0.5ml/min的流速,使用5%Buffer B平衡C4柱50min。
使用Buffer a(超纯水,0.1%三氟乙酸)以及Buffer b(乙腈,0.1%三氟乙酸)按照表1设置线性洗脱梯度。
表1
使用20μl上样体积,在280nm处检测目的蛋白吸光值,根据目的峰面积占比分析目的蛋白纯度,结果如图2所示。
实施例3:投射电子显微镜分析重组蛋白RGD-BH3-HFn重组蛋白质纳米粒的形态
从-80℃冰箱中取出纯化后的自组装蛋白纳米粒,放于37℃水浴锅中快速解冻,待融化后使用166mmol/L碳酸氢钠溶液稀释至0.1mg/ml左右,放于4℃冰盒保存待用。取混匀后的样品滴加到1nm的铜网上,使用纸巾从背面吸干溶液,滴加磷钨酸负染目的蛋白3min,蒸发晾干后,使用HITACHI H-600透射电子显微镜观察目的蛋白纳米粒,并拍照。结果如图3所示。
实施例4:促凋亡铁蛋白纳米粒RGD-BH3-HFn与BH3-HFn抗肿瘤活性测定
所用的C6细胞、bEnd.3细胞培养于含有10%胎牛血清的DMEM高糖培养基中;所用的MCF-7细胞培养于含有10%胎牛血清的1640培养基中;培养基中含有氨苄青霉素和链霉素。
在96孔板接种各自适量的细胞数,C6和MCF-7细胞4000个/孔,bEnd.3细胞12000个/孔,在37℃,5%CO2的恒温箱中培养24h,直至在倒置显微镜下观察细胞贴壁生长并呈现完整细胞形态。更换含有RGD-BH3-HFn与BH3-HFn蛋白的培养基,且保证所有蛋白浓度均为10μmol/L和20μmol/L,并且设置和加入蛋白量相同的碳酸氢钠Buffer对照组。分别与MCF-7、C6、bEnd.3细胞作用48h后,吸出旧培养基,每孔加入新的100μl培养基和10μl CCK-8试剂,在37℃,5%CO2的恒温箱中培养2-3h后,取出使用酶标仪在450nm波长测量其吸光值。抗肿瘤细胞活性结果如图4所示。
流式细胞术定量凋亡实验方法:使用三种细胞,分别在24孔板内加入25-30万个/孔的细胞,进行细胞铺板。在37℃,5%CO2的恒温箱中培养24h,直至在倒置显微镜下观察细胞贴壁生长并呈现完整细胞形态。更换含有活性蛋白的培养基,且保证所有蛋白浓度均为20μmol/L,在37℃,5%CO2的恒温箱中培养24h后,吸出上清,用1x PBS洗涤细胞两次。吸弃PBS,加入不含EDTA的胰酶消化至细胞形态消失细胞变圆,300r/min,离心5min收集细胞。用预冷的1x PBS洗涤细胞,300r/min,离心5min。再次重复洗涤一次。吸弃PBS,加入100μl 1xBinding Buffer,5μl Annexin V-FITC,10μl PI Staining Solution轻轻混匀,避光、室温反应15min后加入400μl 1x Binding Buffer,混匀后放置于冰上。用流式细胞仪分析结果如图5、图6所示。
SEQ ID NO.1
RGD
SEQ ID NO.2
STMGQVGRQLAIIGDDINRRYDSEF
SEQ ID NO.3
MRGDGGSSRSSSTMGQVGRQLAIIGDDINRRYDSEFGGSSRSSTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES
SEQ ID NO.4
atgcgtggtgatggtggtagcagccgtagcagcagcaccatgggtcaagttggtcgtcagctggcaattattggtgatgatattaatcgtcgctatgacagcgaatttggtggtagtagtcgttcaagcaccaccgcaagcaccagccaggttcgtcagaattatcatcaggatagcgaagcagcaattaaccgtcagattaatttggaactgtatgccagctatgtgtatctgagcatgagctattatttcgatcgtgatgatgttgccctgaaaaacttcgcaaaatactttctgcatcagagccatgaagaacgtgaacatgcagaaaaactgatgaaactgcagaatcagcgtggtggtcgtatctttctgcaggatattaagaaaccggattgtgatgattgggaaagcggtctgaatgcaatggaatgtgcactgcatctggaaaaaaatgttaatcagagcctgctggaactgcataaactggcaaccgataaaaatgatccgcatctgtgcgattttatcgaaacccattatctgaacgaacaggtgaaagccattaaagaactgggtgatcatgttaccaatctgcgtaaaatgggtgcaccggaaagtggtctggcagaatacctgtttgataaacataccctgggtgatagcgataacgaaagctaa
SEQ ID NO.5
MSTMGQVGRQLAIIGDDINRRYDSEFGGSSRSSTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYL FDKHTLGDSDNES
SEQ ID NO.6
atgagcaccatgggtcaagttggtcgtcagctggcaattattggtgatgatattaatcgtcgctatgacagcgaatttggtggtagtagtcgttcaagcaccaccgcaagcaccagccaggttcgtcagaattatcatcaggatagcgaagcagcaattaaccgtcagattaatttggaactgtatgccagctatgtgtatctgagcatgagctattatttcgatcgtgatgatgttgccctgaaaaacttcgcaaaatactttctgcatcagagccatgaagaacgtgaacatgcagaaaaactgatgaaactgcagaatcagcgtggtggtcgtatctttctgcaggatattaagaaaccggattgtgatgattgggaaagcggtctgaatgcaatggaatgtgcactgcatctggaaaaaaatgttaatcagagcctgctggaactgcataaactggcaaccgataaaaatgatccgcatctgtgcgattttatcgaaacccattatctgaacgaacaggtgaaagccattaaagaactgggtgatcatgttaccaatctgcgtaaaatgggtgcaccggaaagtggtctggcagaatacctgtttgataaacataccctgggtgatagcgataacgaaagctaa
序列表
<110> 四川大学
<120> 一类促凋亡铁蛋白纳米粒和应用
<160> 6
<170> SIPOSequenceListing 1.0
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Arg Gly Asp
1
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<211> 25
<212> PRT
<213> Escherichia coli
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Ser Thr Met Gly Gln Val Gly Arg Gln Leu Ala Ile Ile Gly Asp Asp
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Ile Asn Arg Arg Tyr Asp Ser Glu Phe
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<210> 3
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<213> Escherichia coli
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Met Arg Gly Asp Gly Gly Ser Ser Arg Ser Ser Ser Thr Met Gly Gln
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Asp Ser Glu Phe Gly Gly Ser Ser Arg Ser Ser Thr Thr Ala Ser Thr
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Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn
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Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met
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Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys
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Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu His Ala Glu Lys Leu
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Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile
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Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu
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Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu
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Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly
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Ser
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atgcgtggtg atggtggtag cagccgtagc agcagcacca tgggtcaagt tggtcgtcag 60
ctggcaatta ttggtgatga tattaatcgt cgctatgaca gcgaatttgg tggtagtagt 120
cgttcaagca ccaccgcaag caccagccag gttcgtcaga attatcatca ggatagcgaa 180
gcagcaatta accgtcagat taatttggaa ctgtatgcca gctatgtgta tctgagcatg 240
agctattatt tcgatcgtga tgatgttgcc ctgaaaaact tcgcaaaata ctttctgcat 300
cagagccatg aagaacgtga acatgcagaa aaactgatga aactgcagaa tcagcgtggt 360
ggtcgtatct ttctgcagga tattaagaaa ccggattgtg atgattggga aagcggtctg 420
aatgcaatgg aatgtgcact gcatctggaa aaaaatgtta atcagagcct gctggaactg 480
cataaactgg caaccgataa aaatgatccg catctgtgcg attttatcga aacccattat 540
ctgaacgaac aggtgaaagc cattaaagaa ctgggtgatc atgttaccaa tctgcgtaaa 600
atgggtgcac cggaaagtgg tctggcagaa tacctgtttg ataaacatac cctgggtgat 660
agcgataacg aaagctaa 678
<210> 5
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<212> PRT
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<210> 6
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gttcgtcaga attatcatca ggatagcgaa gcagcaatta accgtcagat taatttggaa 180
ctgtatgcca gctatgtgta tctgagcatg agctattatt tcgatcgtga tgatgttgcc 240
ctgaaaaact tcgcaaaata ctttctgcat cagagccatg aagaacgtga acatgcagaa 300
aaactgatga aactgcagaa tcagcgtggt ggtcgtatct ttctgcagga tattaagaaa 360
ccggattgtg atgattggga aagcggtctg aatgcaatgg aatgtgcact gcatctggaa 420
aaaaatgtta atcagagcct gctggaactg cataaactgg caaccgataa aaatgatccg 480
catctgtgcg attttatcga aacccattat ctgaacgaac aggtgaaagc cattaaagaa 540
ctgggtgatc atgttaccaa tctgcgtaaa atgggtgcac cggaaagtgg tctggcagaa 600
tacctgtttg ataaacatac cctgggtgat agcgataacg aaagctaa 648
Claims (9)
1.一种促凋亡铁蛋白纳米粒,其特征在于,所述促凋亡铁蛋白纳米粒由RGD肽(SEQ ID
NO.1)、促凋亡BAK蛋白的BH3序列(SEQ ID NO.2)和人铁蛋白的重链亚基(SEQ ID
NO.3)通过连接子形成重组蛋白质。
2.一种DNA分子,其特征在于,编码权利要求1所述的重组蛋白。
3.一种重组质粒,其特征在于,所述重组质粒连接有权利要求2所述的DNA分子。
4.根据权利要求3所述的重组质粒,其特征在于,所述重组质粒选自如下任意一种:pET-21b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、pUC-18以及pUC-19。
5.一种非植物的宿主细胞,其特征在于,所述宿主细胞含有权利要求3或4所述的重组质粒。
6.根据权利要求5所述的宿主细胞,其特征在于,所述宿主细胞为原核细胞或真核细胞,所述宿主细胞选自大肠杆菌、酵母、芽孢杆菌、乳酸杆菌。
7.根据权利要求6所述的宿主细胞,其特征在于,所述细胞为大肠杆菌BL21菌株。
8.根据权利要求1-2任一项所述的不同、权利要求3-4任一项所述的核酸分子或权利要求5-6任意一项所述的表达载体在制备促凋亡铁蛋白纳米粒药物或试剂中的应用。
9.根据权利要求8所述的应用,其特征在于所述的药物用于抗肿瘤治疗,包括包括脑胶质瘤、乳腺癌等。
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