CN109942715A - 一种靶向治疗肿瘤的重组融合蛋白及其制备方法和应用 - Google Patents
一种靶向治疗肿瘤的重组融合蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向治疗肿瘤的重组融合蛋白及其制备方法和应用，属于治疗肿瘤的重组融合蛋白技术领域。本发明中的重组融合蛋白是由穿膜肽、转铁蛋白N端结构域与重组豹蛙抗瘤酶融合而成的重组融合蛋白9R‑rONC‑TFn和9R‑TFn‑rONC，其对应的氨基酸序列分别如序列表中SEQ ID NO:3和SEQ ID NO:4所示。本发明所得重组融合蛋白存在稳定性高和半衰期长等优点，并且具有良好的肿瘤穿透能力和肿瘤靶向作用。
Description
技术领域
本发明属于治疗肿瘤的重组融合蛋白技术领域，具体涉及一种靶向治疗肿瘤的重组融合蛋白及其制备方法和应用。
背景技术
肿瘤是一种多因素、多阶段和多基因变异累积的的复杂疾病，其死亡率在全球范围内高居榜首，虽然全世界每年用于肿瘤治疗的费用高达上万亿美元，但疗效高且特异性强的抗肿瘤药物仍然十分缺乏。一般的肿瘤治疗(化学药物治疗和放射治疗)对快速分裂生长的细胞发挥破坏和杀伤作用，对非肿瘤细胞(分裂期的正常细胞)的毒副作用大，而且长期化疗易产生抗药性，使治疗效果下降。生物制药技术的发展为开发靶向性强且疗效高的抗肿瘤药物提供了良好的发展前景，近年来通过配体-受体介导的主动靶向药物载体系统已经成为抗肿瘤治疗研究的热点(Cheung A,et al.Targeting folate receptor alphafor cancer treatment.Oncotarget,2016.7:52553-74.Marchetti C,et al.Targeteddrug delivery via folate receptors in recurrent ovarian cancer:a review.OncoTargets Ther,2014.7:1223-36)，部分肿瘤患者借助于药物靶向载体治疗的方法提升了生存质量和延长了生存期。抗肿瘤药物偶联载体进行药物的靶向输送作为一种研究方向将越来越受研究者们的青睐。
生理状态下，人体内存在于细胞膜上的受体可特异性地识别配体和抗体分子，并将其导入到体内特定的位置。将配体连接到药物载体表面后，可通过配体-受体介导这一途径将药物特异性地导入体内某一部位，从而实现主动靶向转运。肿瘤组织细胞膜上存在多种特异性受体，如转铁蛋白受体(Transferrin receptor,TFR)、低密度脂蛋白受体和叶酸受体等，有些受体已证实可作为特定肿瘤靶向治疗的靶点。近年来，转铁蛋白(Transferrin,TF)和TFR介导的药物传递系统也取得了令人振奋的结果(Wang X,etal.Targeted delivery of tumor suppressor microRNA-1by transferrin-conjugatedlipopolyplex nanoparticles to patient-derived glioblastoma stem cells.CurrPharm Biotechnol,2014.15:839-46.Lubgan D,Marczak A,Distel L,et al.Transferrinconjugates in the anticancer therapy.Postepy Biochem,2006.52:72-9)。利用TFR有效的靶向作用，TF与抗肿瘤药物的交联物既提高了药物的特异性结合能力，也增强了其治疗效果。TF是由肝脏合成的一种单链糖蛋白，其相对分子质量约75-77kD，体内半衰期约8天(Li H,et al.The role of the transferrin-transferrin-receptor system in drugdelivery and targeting.Trends Pharmacol Sci,2002.23:206-9.)。TF有两个相同的球形结构域，分别位于N-端和C-端，这两个结构域有一定的同源性(Andrés MT,etal.Antimicrobial mechanism of action of transferrins:selective inhibition ofH+-ATPase.Antimicrob Agents Chemother,2010.54:4335-42)。研究者们成功的在毕赤酵母中表达TF的N端结构域蛋白(TFn)，体外实验结果表明该蛋白可以结合并释放铁原子，进一步说明了体外表达的TFn仍具有生物学活性(Mason AB,et al.Production andisolation of the recombinant N-lobe of human serum transferrin from themethylotrophic yeast Pichia pastoris.Protein Expr Purif,1996.8:119-25)。
细胞穿膜肽(Cell-penetrating peptides,CPPs)是一类能携带大分子物质进入细胞的短肽，其穿膜能力不依赖于经典的胞吞作用(Ren J,et al.Development of cell-penetrating peptides as vectors for drug delivery.Acta Pharm Sin,2010,45(1):17-25)。CPPs根据来源可以分为天然存在的和人工合成的两种，天然的细胞穿膜肽来源于天然蛋白质的多肽区域，主要负责将蛋白质向细胞内转运，包括TAT-PTD、Penetration和VP22(Green M,et al.Autonomous functional domains of chemically synthesizedhuman immunodeficiency virus Tat trans-activator protein.Cell,1988,55(6):1179-1188.Frankel AD,et al.Cellular uptake of the tat protein from humanimmunodeficiency virus.Cell,1988,55(6):1189-1193)。这些细胞穿膜肽均为带有正电荷的、长短不等的多肽片段，其中富含精氨酸、赖氨酸等碱性氨基酸残基。利用这一特性，人工合成具有穿膜能力的多聚精氨酸和多聚赖氨酸的穿膜短肽，将生物分子带入细胞内发生作用，是一种细胞渗透性多肽，具有广泛的组织相容性、稳定性、低毒性和低免疫原性、具有特异的组织及细胞递送功能、可人工合成及程序简单等优点(Matsuzaki K,etal.Interactions of an antimicrobial peptide,magainin 2,withipopolysaccharide-containing liposomes as a model for outer membranes ofgram-negative bacteria.FEBS Lett,1999,449(2/3):221-224)。其中9个精氨酸(9R)是一类含有大量正电荷的碱性氨基酸，其作为一个蛋白转运结构域来使用，它的碱性氨基酸残基靠近细胞膜表面，然后CPPs发生旋转使其疏水性氨基酸与细胞膜的疏水核心区相互作用，最后，细胞膜中的磷脂发生轻微断裂，以使CPPs穿透细胞膜(Nakase I,et al.Cell-surface accumulation of flock house virus-derived peptide leads to efficientinternalization via macro pinocytosis,Mol Ther,2009,17(11):1868-1876)。
豹蛙抗瘤酶(Onconase,ONC)是Mikulski等(Mikulski SM,et al.Strikingincrease of survival of mice bearing M109Madison carcinoma treated with anovel protein from amphibian embryos.J Natl Cancer Inst,1990.82:151-3)于1990年在北极豹蛙早期胚胎中首次发现的，Ardelt等(Ardelt W,et al.Onconase andamphinase,the antitumor ribonucleases from Rana pipiens oocytes.Curr PharmBiotechnol,2008.9:215-25)测出了其全部序列，并根据oncology和ribonuclease将它命名为Onconase。ONC由104个氨基酸组成，分子量为11.8kD，是RNase A家族中最小的单结构域蛋白，含2个β折叠片层结构和3个α螺旋结构。ONC是一种新型的抗癌药物，对多种实体瘤有很强的杀伤作用，是第一个进入抗肿瘤临床试验的核糖核酸酶，也是当前全球重点研究的100种新药之一(Costanzi J SD,et al.Ribonucleases as a novel pro-apoptoticanticancer strategy:Review of the preclinical and clinical data forranpirnase.Cancer Invest,2005.23:643-50)。ONC在有效作用剂量下，主要作用于对数生长期的肿瘤细胞，而对正常细胞没有明显作用(Grabarek J,et al.Activation ofcaspases and serine proteases during apoptosis induced by onconase(Ranpirnase).Exp Cell Res,2002.278:61-71)，同时，它还具有机体不良反应少、免疫原性低、不易产生耐药性等优点，是一种极具潜力的抗肿瘤药物。
ONC进入细胞的第一步是结合在细胞表面。但至今尚未发现细胞表面存在ONC受体，ONC结合到细胞表面后，通过内吞作用进入细胞质，然后被运送至核内体。内吞是ONC细胞毒性的限制因素。为了提高ONC进入肿瘤细胞的效率以及靶向杀伤肿瘤细胞的能力，本发明将其与9R和TFn连接成融合蛋白的形式来提高ONC的靶向性、穿透能力和抗肿瘤活性，为以后ONC的靶向治疗肿瘤提供了重要的技术帮助。
发明内容
本发明解决的技术问题是提供了一种靶向治疗肿瘤的重组融合蛋白及其制备方法和应用，该重组蛋白由重组豹蛙抗瘤酶(rONC)和转铁蛋白N端结构域(TFn)及9个精氨酸的穿膜肽(9R)融合而成，所得重组融合蛋白存在稳定性高和半衰期长等优点，并且具有良好的肿瘤穿透能力和肿瘤靶向作用。
本发明为解决上述技术问题采用如下技术方案，一种靶向治疗肿瘤的重组融合蛋白，其特征在于：所述重组融合蛋白是由穿膜肽、转铁蛋白N端结构域与重组豹蛙抗瘤酶融合而成的重组融合蛋白9R-rONC-TFn和9R-TFn-rONC，其对应的氨基酸序列分别如序列表中SEQ ID NO:3和SEQ ID NO:4所示。
本发明所述的靶向治疗肿瘤的重组融合蛋白编码基因，其特征在于：所述重组融合蛋白9R-rONC-TFn和9R-TFn-rONC编码基因的核苷酸序列分别如序列表中SEQ ID NO:1和SEQ ID NO:2所示。
优选的，所述靶向治疗肿瘤的重组融合蛋白编码基因由重组豹蛙抗瘤酶核苷酸序列与穿膜肽和转铁蛋白N端结构域基因片段通过连接肽连接而成。
优选的，所述连接肽为GS linker((Gly4Ser)3)。
本发明所述的含有靶向治疗肿瘤的重组融合蛋白编码基因的表达载体和表达载体的宿主细胞，其特征在于：所述表达载体为pET27b(+)，宿主细胞为Rossetta(DE3)。
本发明所述的靶向治疗肿瘤的重组融合蛋白的制备方法，其特征在于具体过程为：将编码重组融合蛋白9R-rONC-TFn和9R-TFn-rONC的两种核苷酸序列分别与表达载体相连接得到重组表达载体；再将重组表达载体分别转化宿主细胞；分别筛选高表达阳性宿主细胞，分别培养细胞并诱导表达重组融合蛋白，收集菌体、破碎、离心、变性、复性、纯化得到靶向治疗肿瘤的重组融合蛋白。
优选的，将重组表达载体分别转化表达菌株Rosseta(DE3)，转化后的单菌落分别接种至20mL含50μg/mL Kan的LB培养基中，于37℃培养8h，以体积比1:100接种于另一20mL含50μg/mL Kan的LB培养基中，于37℃培养，当A600在0.35时，在温度为37℃、IPTG终浓度为0.25mmol/L、转速为80r/min的条件下诱导6h，在此条件下进行表达重组融合蛋白9R-rONC-TFn和9R-TFn-rONC时，能够显著提高重组融合蛋白的表达水平。
优选的，将重组表达载体分别转化宿主细胞，筛选高表达阳性克隆，培养并诱导表达重组融合蛋白9R-rONC-TFn和9R-TFn-rONC，通过中空纤维柱膜过滤技术对菌体进行富集，破碎，离心，上清液调pH值至6.0利用SP Sepharose 6FF亲和层析，Superdex 75凝胶过滤层析分离纯化得到靶向治疗肿瘤的重组融合蛋白。
本发明所述的靶向治疗肿瘤的重组融合蛋白作为靶向治疗肿瘤药物的用途，靶向治疗肿瘤的重组融合蛋白作为靶向治疗肿瘤药物的稳定性高且半衰期长，具有良好的肿瘤细胞穿透能力和肿瘤细胞靶向作用，并且活性显著提高。
本发明与现有技术相比具有以下有益效果：本发明通过细胞试验结果表明，本发明制得的重组融合蛋白9R-rONC-TFn和9R-TFn-rONC相比于ONC，活性显著提高，能够更加有效地杀伤多种肿瘤细胞，特别是重组融合蛋白9R-rONC-TFn，其杀伤效果更佳。本发明的重组融合蛋白可作为靶向治疗肿瘤的药物。
附图说明
图1是重组融合蛋白9R-rONC-TFn和9R-TFn-rONC在大肠杆菌中表达的SDS-PAGE电泳分析；
图2是纯化后重组融合蛋白9R-rONC-TFn和9R-TFn-rONC的SDS-PAGE电泳分析；
图3是重组融合蛋白9R-rONC-TFn和9R-TFn-rONC结合TF受体能力检测；
图4是重组融合蛋白9R-rONC-TFn和9R-TFn-rONC的体外细胞活性检测；
图5是重组融合蛋白9R-rONC-TFn和9R-TFn-rONC与TF竞争结合肿瘤细胞的能力检测。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
说明：本发明中涉及的基因的设计、合成和克隆、表达载体的构建、核酸提取、测序和鉴定，以及表达产物的分离和纯化等操作步骤，可按照本领域已知的技术进行(参见CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1
重组融合蛋白9R-rONC-TFn和9R-TFn-rONC表达载体的构建
根据大肠杆菌密码子偏好性，设计出2种基因，其核苷酸序列分别如序列表中SEQID NO:1(9R-rONC-TFn)和SEQ ID NO:2(9R-TFn-rONC)所示。将这2种基因送至上海捷瑞生物公司合成，同时在各基因两端设计Nco I与BamH I酶切位点。
将2种合成的含有各自目的基因片段的载体和表达载体pET27b(+)，分别用Nco I与BamH I双酶切，酶切完毕后，胶回收各自需要的目标片段。使用T4DNA连接酶将2种目的片段分别与原核表达载体pET27b(+)连接，连接反应体系为10μL，混匀，4℃连接过夜，然后各自转化至大肠杆菌DH5a中。挑取阳性克隆，经过酶切鉴定后，即分别构建得到2种重组质粒pET27b-9R-rONC-TFn和pET27b-9R-TFn-rONC。
实施例2
重组融合蛋白9R-rONC-TFn和9R-TFn-rONC的表达及纯化
(1)转化、培养并诱导表达
将含有正确序列的2种重组质粒pET27b-9R-rONC-TFn和pET27b-9R-TFn-rONC分别转化至表达菌株Rosseta(DE3)(北京全式金生物技术有限公司,目录号：CD801)。转化后的单菌落分别接种至20mL含Kan(50ug/mL)的LB培养基中，于37℃培养8h，以1:100接种于另一20mL含Kan(50ug/mL)的LB培养基中，于37℃培养，当A600在0.35左右时，加入IPTG至终浓度为0.25mmol/L进行诱导，诱导温度为37℃，5h后收获菌体，用Binding buffer(20mmol/LNa3PO4,pH 7.0)重悬菌体，破碎菌体后离心，分别取上清和沉淀进行12％SDS-PAGE电泳分析。结果显示9R-rONC-TFn和9R-TFn-rONC的重组融合蛋白以包涵体形式表达，如图1所示，泳道1：蛋白标准分子量Marker；2：未诱导的全菌；3、4、5：9R-rONC-TFn诱导后全菌、菌体沉淀、菌体上清；6、7、8：9R-TFn-rONC诱导后全菌、菌体沉淀、菌体上清。
(2)蛋白纯化
向菌体中加入一定浓度溶菌酶(1mg/mL)，冰上放置30min，超声波细胞破碎菌体细胞(工作1s，间隔1s，4min/次，共3次循环)。菌体破碎彻底后，利用QuixStand预处理系统(750kD超滤中空纤维柱)处理细胞破碎液，富集包涵体，弃去膜透过端液体。当总体积约为60mL时，加入100mL wash buffer(20mmol/L Tris，2mol/L Urea，150mmol/LNaCl，pH 8.0)洗涤包涵体。当溶液体积为50mL，再向其中加入洗涤液100mL，重复上述实验4次。
洗涤完毕后，当溶液体积为50mL，关闭透过端，向洗涤后的包涵体中加入150mL的变性液(20mmol/L Tris，10mol/L Urea，150mmol/L NaCl，pH 8.0)，循环变性2h。打开透过端，膜透过端收集液即为9R-rONC-TFn或9R-TFn-rONC变性液。用5KD中空纤维柱对变性后的9R-rONC-TFn或9R-TFn-rONC进行浓缩，至体积80mL后进行复性，将装有复性液(20mmol/LTris，50mmol/L NaCl，pH 8.0)的容器用胶皮管与中空纤维柱的储液器连接。储液器密封后，透过端流出液体后，由于储存器中产生负压，使复性液以一定的速度滴加至变性液中，缓慢匀速复性。当加入复性液体积为变性液6倍时，即复性完毕，复性后溶液调pH至6.0，8000rpm/min，4℃离心20min，收集上清。
上清液过0.22μm滤膜澄清后，通过泵进入AKTA purifier 100系统，与2-3倍柱体积Binding buffer(PBS，pH 6.0)平衡好的SP Sepharose 6FF柱(装于XK16/20空柱，柱高10cm，流速100cm/h)完全结合后，用4-5倍柱体积的binding buffer冲洗杂蛋白；当紫外曲线达到稳定的基线时，用Wash buffer(20mmol/L Na3PO4，200mmol/L NaCl，pH 7.0)冲洗至基线稳定，再用2-3倍柱体积Elution buffer(20mmol/L Na3PO4，500mmol/L NaCl，pH 7.0)洗脱目的蛋白，把结合在填料上的融合蛋白洗脱下来并收集到试管中。
把Superdex 75凝胶过滤柱(装于Column XK26/70空柱中，柱体积340mL，流速2mL/min)接到AKTA purifier 100系统中，先用2倍柱体积的蒸馏水替换其保护液(体积分数20％乙醇)，再用2倍柱体积的Desalting buffer(20mmol/L Na3PO4，150mmol/L NaCl，pH7.0)平衡柱子，然后将亲和层析洗脱液通过Superloop进样。收集各洗脱峰，并进行15％SDS-PAGE电泳分析，结果显示经纯化过后，四种融合蛋白纯度在95％以上，如图2所示，泳道1：蛋白标准分子量Marker；2：纯化后的9R-rONC-TFn融合蛋白；3：纯化后的9R-TFn-rONC融合蛋白。
实施例3
重组融合蛋白9R-rONC-TFn和9R-TFn-rONC结合TF受体(TFR)检测
将ELISA板每孔加100μL不同浓度重组融合蛋白9R-rONC-TFn或9R-TFn-rONC(浓度：500μg/mL，100μg/mL，20μg/mL，4μg/mL，0.8μg/mL，稀释液为0.1M NaCO3-NaHCO3，pH9.6)，4℃下包被过夜过夜。所有后续步骤均在37℃下进行。用PBST(0.05wt％吐温-20，PBS)洗涤三次，用300μL5wt％脱脂奶粉PBS封闭2h。每孔加入100μL(0.1μg/mL)带His标签的TFR(Sino Biological)，孵育1h，用PBST洗涤3次，加入100μL鼠源抗His抗体(1:500稀释)，再孵育1h。用PBST洗涤3次平板后，加入100μL山羊抗鼠IgG-HRP抗体(1:7500稀释)，孵育1h，用PBST洗涤5次平板后，加入100μL TMB溶液。用50μL的1M硫酸溶液终止反应。用酶标仪在450nm处测量吸光度。
结果如图3所示，重组融合蛋白9R-rONC-TFn或9R-TFn-rONC与TFR的结合呈剂量依赖性，这说明两种重组融合蛋白都具有靶向结合TF受体的能力。
实施例4
重组融合蛋白9R-rONC-TFn和9R-TFn-rONC的体外细胞活性检测
分别将HepG2、Hela和L02细胞接种于96孔板，每孔5000个细胞，10wt％FBS+90wt％1640培养至对数生长期。分别向三种细胞中加入不同浓度rONC、9R-rONC-TFn和9R-TFn-rONC，设置无药物的阴性对照组和仅含培养基的调零组。药物处理细胞后48h，加入MTT，继续培养4h，吸取上清后，每孔加入150μL DMSO待完全溶解后，读取570nm下OD值。计算细胞增殖抑制率，细胞增殖抑制率％＝(1-实验组A570nm/对照组A570nm)×100％。
结果如图4所示，三种蛋白对HepG2和Hela细胞有杀伤作用，对L02正常细胞没有作用，重组融合蛋白9R-rONC-TFn和9R-TFn-rONC对肿瘤细胞的杀伤作用强于rONC，特别是重组融合蛋白9R-rONC-TFn，其杀伤肿瘤细胞的能力更强。
实施例5
重组融合蛋白9R-rONC-TFn和9R-TFn-rONC与TF竞争结合肿瘤细胞检测
将对数生长期的HepG2细胞稀释为5×104/mL，分别接种于96孔板，200μL/孔。培养24h后弃去培养液，竞争实验时先用1μM TF与细胞共培养30min，然后每孔加9R-rONC-TFn、9R-TFn-rONC或rONC的培养液，使9R-rONC-TFn、9R-TFn-rONC和rONC的终浓度依次为0.05nM、0.2nM、0.8nM、3.2nM、12.8nM、50nM、200nM、800nM和3200nM，每个浓度3个复孔。培养72h后，每孔加入5mg/mL的MTT 20μL。继续培养4h，轻轻吸去上清，加入150μL DMSO溶解，用酶标仪读取570nm处测定OD值。
结果如图5所示，TF对rONC杀伤肿瘤细胞没有影响，但是对重组融合蛋白9R-rONC-TFn和9R-TFn-rONC杀伤肿瘤细胞有竞争抑制作用，这说明重组融合蛋白9R-rONC-TFn和9R-TFn-rONC能够靶向结合肿瘤细胞表面。
以上实施例描述了本发明的基本原理、主要特征及优点，本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明原理的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发明保护的范围内。
序列表
<110> 河南师范大学
<120> 一种靶向治疗肿瘤的重组融合蛋白及其制备方法和应用
<130> 2019
<160> 4
<170> SIPOSequenceListing 1.0
<210> 5
<211> 1446
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agaaggcgta gaagaaggag aaggagaggt ggcggtggct ccggcggtgg tgggtcgggt 60
ggcggcggat ctcaagactg gttgaccttc caaaagaagc acatcaccaa caccagagac 120
gtcgactgtg acaacatcat gtccaccaac ttgttccact gtaaggacaa gaacaccttc 180
atctactccc gtccagagcc agtcaaggct atctgcaagg gtatcatcgc ttccaagaac 240
gtccttacca cctccgagtt ctacttgtcc gactgtaacg ttaccagtag accatgcaag 300
tacaagttga agaagtccac caacaagttc tgcgtcacct gtgagaacca agctccagtt 360
cacttcgttg gcgtcggtag ttgtggtggc ggtggctccg gcggtggtgg gtcgggtggc 420
ggcggatctg tccctgataa aactgtgaga tggtgtgcag tgtcggagca tgaggccact 480
aagtgccaga gtttccgcga ccatatgaaa agcgtcattc catccgatgg tcccagtgtt 540
gcttgtgtga agaaagcctc ctaccttgat tgcatcaggg ccattgcggc aaacgaagcg 600
gatgctgtga cactggatgc aggtttggtg tatgatgctt acctggctcc caataacctg 660
aagcctgtgg tggcagagtt ctatgggtca aaagaggatc cacagacttt ctattatgct 720
gttgctgtgg tgaagaagga tagtggcttc cagatgaacc agcttcgagg caagaagtcc 780
tgccacacgg gtctaggcag gtccgctggg tggaacatcc ccataggctt actttactgt 840
gacttacctg agccacgtaa acctcttgag aaagcagtgg ccaatttctt ctcgggcagc 900
tgtgcccctt gtgcggatgg gacggacttc ccccagctgt gtcaactgtg tccagggtgt 960
ggctgctcca cccttaacca atacttcggc tactcgggag ccttcaagtg tctgaaggat 1020
ggtgctgggg atgtggcctt tgtcaagcac tcgactatat ttgagaactt ggcaaacaag 1080
gctgacaggg accagtatga gctgctttgc ctggacaaca cccggaagcc ggtagatgaa 1140
tacaaggact gccacttggc ccaggtccct tctcataccg tcgtggcccg aagtatgggc 1200
ggcaaggagg acttgatctg ggagcttctc aaccaggccc aggaacattt tggcaaagac 1260
aaatcaaaag aatttcaact attcagctct cctcatggga aggacctgct gtttaaggac 1320
tctgcccacg ggtttttaaa agtccccccc aggatggatg ccaagatgta cctgggctat 1380
gagtatgtca ctgccatccg gaatctacgg gaaggcacat gcccagaagc cccaacagat 1440
gaataa 1446
<210> 6
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<212> DNA
<213> 人工序列(Artificial Sequence)
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agaaggcgta gaagaaggag aaggagaggt ggcggtggct ccggcggtgg tgggtcgggt 60
ggcggcggat ctgtccctga taaaactgtg agatggtgtg cagtgtcgga gcatgaggcc 120
actaagtgcc agagtttccg cgaccatatg aaaagcgtca ttccatccga tggtcccagt 180
gttgcttgtg tgaagaaagc ctcctacctt gattgcatca gggccattgc ggcaaacgaa 240
gcggatgctg tgacactgga tgcaggtttg gtgtatgatg cttacctggc tcccaataac 300
ctgaagcctg tggtggcaga gttctatggg tcaaaagagg atccacagac tttctattat 360
gctgttgctg tggtgaagaa ggatagtggc ttccagatga accagcttcg aggcaagaag 420
tcctgccaca cgggtctagg caggtccgct gggtggaaca tccccatagg cttactttac 480
tgtgacttac ctgagccacg taaacctctt gagaaagcag tggccaattt cttctcgggc 540
agctgtgccc cttgtgcgga tgggacggac ttcccccagc tgtgtcaact gtgtccaggg 600
tgtggctgct ccacccttaa ccaatacttc ggctactcgg gagccttcaa gtgtctgaag 660
gatggtgctg gggatgtggc ctttgtcaag cactcgacta tatttgagaa cttggcaaac 720
aaggctgaca gggaccagta tgagctgctt tgcctggaca acacccggaa gccggtagat 780
gaatacaagg actgccactt ggcccaggtc ccttctcata ccgtcgtggc ccgaagtatg 840
ggcggcaagg aggacttgat ctgggagctt ctcaaccagg cccaggaaca ttttggcaaa 900
gacaaatcaa aagaatttca actattcagc tctcctcatg ggaaggacct gctgtttaag 960
gactctgccc acgggttttt aaaagtcccc cccaggatgg atgccaagat gtacctgggc 1020
tatgagtatg tcactgccat ccggaatcta cgggaaggca catgcccaga agccccaaca 1080
gatgaaggtg gcggtggctc cggcggtggt gggtcgggtg gcggcggatc tcaagactgg 1140
ttgaccttcc aaaagaagca catcaccaac accagagacg tcgactgtga caacatcatg 1200
tccaccaact tgttccactg taaggacaag aacaccttca tctactcccg tccagagcca 1260
gtcaaggcta tctgcaaggg tatcatcgct tccaagaacg tccttaccac ctccgagttc 1320
tacttgtccg actgtaacgt taccagtaga ccatgcaagt acaagttgaa gaagtccacc 1380
aacaagttct gcgtcacctg tgagaaccaa gctccagttc acttcgttgg cgtcggtagt 1440
tgttaa 1446
<210> 7
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Ser Glu Gln Ile Asp Asn Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp
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Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp
35 40 45
Cys Asp Asn Ile Met Glu Thr Ser Thr Asn Leu Phe His Cys Lys Asp
50 55 60
Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys
65 70 75 80
Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr
85 90 95
Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys
100 105 110
Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val
115 120 125
His Phe Val Gly Val Gly Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Val Pro Asp Lys Thr Val Arg Trp Cys
145 150 155 160
Ala Val Ser Glu His Glu Ala Thr Lys Cys Gln Ser Phe Arg Asp His
165 170 175
Met Glu Thr Lys Ser Val Ile Pro Ser Asp Gly Pro Ser Val Ala Cys
180 185 190
Val Lys Lys Ala Ser Tyr Leu Asp Cys Ile Arg Ala Ile Ala Ala Asn
195 200 205
Glu Ala Asp Ala Val Thr Leu Asp Ala Gly Leu Val Tyr Asp Ala Tyr
210 215 220
Leu Ala Pro Asn Asn Leu Lys Pro Val Val Ala Glu Phe Tyr Gly Ser
225 230 235 240
Lys Glu Asp Pro Gln Thr Phe Tyr Tyr Ala Val Ala Val Val Lys Lys
245 250 255
Asp Ser Gly Phe Gln Met Glu Thr Asn Gln Leu Arg Gly Lys Lys Ser
260 265 270
Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Asn Ile Pro Ile Gly
275 280 285
Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg Lys Pro Leu Glu Lys Ala
290 295 300
Val Ala Asn Phe Phe Ser Gly Ser Cys Ala Pro Cys Ala Asp Gly Thr
305 310 315 320
Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro Gly Cys Gly Cys Ser Thr
325 330 335
Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala Phe Lys Cys Leu Lys Asp
340 345 350
Gly Ala Gly Asp Val Ala Phe Val Lys His Ser Thr Ile Phe Glu Asn
355 360 365
Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr Glu Leu Leu Cys Leu Asp
370 375 380
Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys Asp Cys His Leu Ala Gln
385 390 395 400
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405 410 415
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420 425 430
Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser Pro His Gly Lys
435 440 445
Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu Lys Val Pro Pro
450 455 460
Arg Met Glu Thr Asp Ala Lys Met Glu Thr Tyr Leu Gly Tyr Glu Tyr
465 470 475 480
Val Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro
485 490 495
Thr Asp Glu
<210> 8
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Ser Glu Gln Ile Asp Asn Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Pro
20 25 30
Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu Ala Thr Lys
35 40 45
Cys Gln Ser Phe Arg Asp His Met Glu Thr Lys Ser Val Ile Pro Ser
50 55 60
Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp Cys
65 70 75 80
Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp Ala
85 90 95
Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro Val
100 105 110
Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr Tyr
115 120 125
Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Glu Thr Asn
130 135 140
Gln Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala
145 150 155 160
Gly Trp Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro
165 170 175
Arg Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys
180 185 190
Ala Pro Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys
195 200 205
Pro Gly Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly
210 215 220
Ala Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys
225 230 235 240
His Ser Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln
245 250 255
Tyr Glu Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr
260 265 270
Lys Asp Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala Arg
275 280 285
Ser Met Glu Thr Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn
290 295 300
Gln Ala Gln Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu
305 310 315 320
Phe Ser Ser Pro His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His
325 330 335
Gly Phe Leu Lys Val Pro Pro Arg Met Glu Thr Asp Ala Lys Met Glu
340 345 350
Thr Tyr Leu Gly Tyr Glu Tyr Val Thr Ala Ile Arg Asn Leu Arg Glu
355 360 365
Gly Thr Cys Pro Glu Ala Pro Thr Asp Glu Gly Gly Gly Gly Ser Gly
370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Trp Leu Thr Phe Gln
385 390 395 400
Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met
405 410 415
Glu Thr Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile
420 425 430
Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala
435 440 445
Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn
450 455 460
Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys
465 470 475 480
Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val
485 490 495
Gly Ser Cys
Claims (9)
1.一种靶向治疗肿瘤的重组融合蛋白，其特征在于：所述重组融合蛋白是由穿膜肽、转铁蛋白N端结构域与重组豹蛙抗瘤酶融合而成的重组融合蛋白9R-rONC-TFn和9R-TFn-rONC，其对应的氨基酸序列分别如序列表中SEQ ID NO:3和SEQ ID NO:4所示。
2.一种权利要求1所述的靶向治疗肿瘤的重组融合蛋白编码基因，其特征在于：所述重组融合蛋白9R-rONC-TFn和9R-TFn-rONC编码基因的核苷酸序列分别如序列表中SEQ IDNO:1和SEQ ID NO:2所示。
3.根据权利要求2所述的靶向治疗肿瘤的重组融合蛋白编码基因，其特征在于：所述靶向治疗肿瘤的重组融合蛋白编码基因由重组豹蛙抗瘤酶核苷酸序列与穿膜肽和转铁蛋白N端结构域基因片段通过连接肽连接而成。
4.根据权利要求3所述的靶向治疗肿瘤的重组融合蛋白编码基因，其特征在于：所述连接肽为GS linker((Gly4Ser)3)。
5.一种含有权利要求2或3所述的靶向治疗肿瘤的重组融合蛋白编码基因的表达载体和表达载体的宿主细胞，其特征在于：所述表达载体为pET27b(+)，宿主细胞为Rossetta(DE3)。
6.一种权利要求1所述的靶向治疗肿瘤的重组融合蛋白的制备方法，其特征在于具体过程为：将编码重组融合蛋白9R-rONC-TFn和9R-TFn-rONC的两种核苷酸序列分别与表达载体相连接得到重组表达载体；再将重组表达载体分别转化宿主细胞；分别筛选高表达阳性宿主细胞，分别培养细胞并诱导表达重组融合蛋白，收集菌体、破碎、离心、变性、复性、纯化得到靶向治疗肿瘤的重组融合蛋白。
7.根据权利要求6所述的靶向治疗肿瘤的重组融合蛋白的制备方法，其特征在于：将重组表达载体分别转化表达菌株Rosseta(DE3)，转化后的单菌落分别接种至20mL含50μg/mLKan的LB培养基中，于37℃培养8h，以体积比1:100接种于另一20mL含50μg/mL Kan的LB培养基中，于37℃培养，当A600在0.35时，在温度为37℃、IPTG终浓度为0.25mmol/L、转速为80r/min的条件下诱导6h，在此条件下进行表达重组融合蛋白9R-rONC-TFn和9R-TFn-rONC时，能够显著提高重组融合蛋白的表达水平。
8.根据权利要求6所述的靶向治疗肿瘤的重组融合蛋白的制备方法，其特征在于：将重组表达载体分别转化宿主细胞，筛选高表达阳性克隆，培养并诱导表达重组融合蛋白9R-rONC-TFn和9R-TFn-rONC，通过中空纤维柱膜过滤技术对菌体进行富集，破碎，离心，上清液调pH值至6.0利用SP Sepharose 6FF亲和层析，Superdex 75凝胶过滤层析分离纯化得到靶向治疗肿瘤的重组融合蛋白。
9.权利要求1所述的靶向治疗肿瘤的重组融合蛋白作为靶向治疗肿瘤药物的用途，该靶向治疗肿瘤的重组融合蛋白作为靶向治疗肿瘤药物的稳定性高且半衰期长，具有良好的肿瘤细胞穿透能力和肿瘤细胞靶向作用，并且活性显著提高。
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