CN111909274A - 一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法 - Google Patents

一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法 Download PDF

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CN111909274A
CN111909274A CN202010705668.1A CN202010705668A CN111909274A CN 111909274 A CN111909274 A CN 111909274A CN 202010705668 A CN202010705668 A CN 202010705668A CN 111909274 A CN111909274 A CN 111909274A
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王文义
徐畅
姜长安
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Abstract

本发明涉及一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法,同时还涉及该纳米抗体的氨基酸序列、基因编码序列以及能够表达该纳米抗体的表达载体及宿主细胞。本发明提供的GPC3的纳米抗体的氨基酸序列如SEQ ID NO:7所示,编码基因的核苷酸序列如SEQ ID NO:8所示。本发明涉及的GPC3的纳米抗体能够通过结合细胞膜表达的GPC3,特异性地识别GPC3高表达的肝癌细胞。该抗体具有突出高稳定性,可用于GPC3的功能研究,也可用于肝细胞癌的诊断试剂和治疗药物的开发。

Description

一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体 及其制备方法
技术领域
本发明涉及一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体及其制备方法,还涉及编码其的基因、表达载体、宿主细胞及相关应用,属于生物技术领域。
背景技术
肝癌素有“癌症之王”之名,是死亡率高居第三的癌症。中国的肝癌新发病例数及死亡病例数占全球总数的一半以上。肝细胞癌(Hepatocellular carcinoma,HCC)是肝癌的最主要形式,约占75%。由于肝癌早期症状隐匿以及诊断手段的限制,多数肝癌患者被确诊时已处于肝癌晚期,而晚期肝癌预后差,治疗十分困难。因此,肝癌的早期诊断对于提高肝癌患者的生存期意义重大。而靶向药物由于其靶向性强、副作用小,成为肝癌诊断和治疗最具前景的药物之一。肝癌肿瘤细胞的标志蛋白是靶向药物常用的分子靶标。
磷脂酰肌醇蛋白聚糖3(Glypican-3,GPC3)是肝细胞癌的标志蛋白,在肝细胞癌肿瘤细胞中特异性高表达。GPC3是一种通过糖基磷脂酰肌醇(Glycosyl phosphatidylinositol,GPI)锚定在细胞膜上的硫酸乙酰肝素蛋白多糖,参与调控个体发育、细胞增殖和分化等过程。正常情况下,GPC3仅在胎儿肝脏中表达,健康成人肝脏中一般无GPC3表达或表达量较低。然而在多数肝细胞癌患者的肝脏组织中,GPC3呈过量表达现象。研究显示,GPC3作为肝细胞癌标志蛋白的灵敏度为70-94%,特异性为86-100%。因此,GPC3是肝细胞癌靶向药物的良好靶标分子。靶标分子的特异性抗体在靶向药物中发挥靶向作用,将药物特异性的结合至分子靶标,发挥诊断和(或)治疗功能。目前,已有特异性识别GPC3的单克隆抗体的报道,然而鉴于传统抗体稳定性低、制备工艺复杂及成本昂贵等局限性,目前迫切需要开发针对GPC3的新型抗体。
纳米抗体(Nanobody,Nb)作为一种优质新型抗体,是骆驼科及软骨鱼类血清中存在的一种天然缺失轻链的重链抗体(Heavy-chain-only antibodies,HcAbs)的重链可变区(Variable domain of the heavy chain of HcAbs,VHH)部分。纳米抗体具有完整的抗原结合能力,为最小的抗原结合片段,分子量为13-15kDa,大小(晶体结构直径为2.5纳米,长度为4纳米)位于纳米尺寸。纳米抗体结构主要分为保守的骨架区(Framework regions,FRs)和序列多变的互补决定区(Complementarity determining regions,CDRs),分别负责维持纳米抗体的基本结构和决定与抗原的特异性结合。CDR根据其在整个抗体中的位置不同,分为三个独立区域,即互补决定区1(CDR1)、互补决定区2(CDR2)和互补决定区3(CDR3)。与传统抗体相比,纳米抗体体积更小,可以更深入的穿透癌变组织,识别隐藏抗原位点。除此之外,纳米抗体具有特异性强,分子量小,溶解性高,结构稳定性高,与抗原结合力强,组织穿透能力强,人体免疫原性低,易筛选,易制备等诸多优点,可应用于疾病的研究、诊断和治疗中。分子成像技术由于其非侵入性和高分辨率的特点颇受关注,纳米抗体是分子成像中优质的靶向分子,它可以迅速定位于靶标位点,并且游离的纳米抗体能通过肾脏迅速排出体外,从而保证了特异性信号较高的信噪比,有利于获得高分辨率的肿瘤解剖学影像及靶标蛋白分子定位信息,有效提高癌症的早期诊断效率。在肿瘤治疗中,纳米抗体与肿瘤药物进行偶联,可用于靶向输送药物;在嵌合抗原受体T细胞免疫疗法(Chimeric AntigenReceptor T-Cell Immunotherapy,CAR-T)中,纳米抗体作为靶向分子可将T细胞精准靶向肿瘤细胞,从而高效、精准的杀灭肿瘤细胞。特别地,相比于传统抗体,纳米抗体由于其具有强组织穿透能力,在HCC等实体瘤相关疾病中更具应用前景。除此之外,该纳米抗体也可对GPC3蛋白进行靶向示踪及功能操纵等,用于GPC3相关的生物医学研究。
发明内容
本发明所要解决的技术问题是提供一种GPC3的纳米抗体及其制备方法,为肝细胞癌的诊断和治疗提供有效的靶向分子,同时还提供编码该纳米抗体的基因、表达载体、宿主细胞及相关应用。
本发明解决上述技术问题的一种技术方案如下:一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体,所述纳米抗体包括3个互补决定区,分别为CDR1、CDR2和CDR3;其中,CDR1的氨基酸序列如SEQ ID NO.1所示(Ser Ser Asp Phe Ser Arg Asn Ala),CDR2的氨基酸序列由SEQ ID NO.2所示(Asn Trp Thr Gly Ser Gly Tyr Ala),CDR3的氨基酸序列如SEQ ID NO.3所示(Ala Pro Leu Ala Cys Ser Asn Cys Phe Pro Tyr Lys His TyrVal Tyr)。
在上述技术方案的基础上,本发明还可以做如下改进。
进一步,所述纳米抗体还包括与三个互补决定区交替连接的四个骨架区,分别为FR1、FR2、FR3和FR4;其中,FR1的氨基酸序列如SEQ ID NO.13所示(Gln Val Gln Leu ValGlu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala AlaSer),FR2的氨基酸序列由SEQ ID NO.14所示(Leu Arg Trp Tyr Arg Gln Ala Pro GlyLys Glu Arg Glu Trp Val Cys Gly Ile),FR3的氨基酸序列如SEQ ID NO.15所示(TyrGlu Asp Ser Val Lys Gly Arg Phe Thr Cys Ser Arg Asp Asp Ala Arg Asn Thr ValTyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys),FR4的氨基酸序列如SEQ ID NO.16所示(Trp Gly Gln Gly Thr Gln Val Thr ValSer Ser)。
进一步,所述纳米抗体的氨基酸序列如SEQ ID NO:7所示(Gln Val Gln Leu ValGlu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala AlaSer Ser Ser Asp Phe Ser Arg Asn Ala Leu Arg Trp Tyr Arg Gln Ala Pro Gly LysGlu Arg Glu Trp Val Cys Gly Ile Asn Trp Thr Gly Ser Gly Tyr Ala Tyr Glu AspSer Val Lys Gly Arg Phe Thr Cys Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr LeuGln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Pro Leu AlaCys Ser Asn Cys Phe Pro Tyr Lys His Tyr Val Tyr Trp Gly Gln Gly Thr Gln ValThr Val Ser Ser)。
本发明解决上述技术问题的另一种技术方案如下:一种编码所述具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体的基因,包括三段分别编码其3个互补决定区的核苷酸序列,其中,编码CDR1的核苷酸序列如SEQ ID NO.4所示(tcgagcgatt tttcgcgtaacgcc),编码CDR2的核苷酸序列如SEQID NO.5所示(aactggaccg gttctggcta cgcg),编码CDR3的核苷酸序列如SEQ ID NO.6所示(gcgccgctgg cttgttctaa ttgttttccgtataagcattatgtttac)。
进一步,所述基因的核苷酸序列如SEQ ID NO:8所示(caagttcaat tagtcgagtccggcggagct ctggtccagc ctggaggtag tctgcgttta tcctgcgcag ccagctcgag cgatttttcgcgtaacgccc tccgctggta tcgccaggca ccgggtaagg agcgcgaatg ggtatgcggt attaactggaccggttctgg ctacgcgtac gaagacagcg ttaaagggcg ttttacttgt tcccgcgacg acgctcgtaacacagtctat ttacaattaa actcattaaa gcctgaagac acagcggtat attactgcgc gccgctggcttgttctaatt gttttccgta taagcattat gtttactggg ggcagggcac gcaggtaacc gttagctca)。
本发明解决上述技术问题的另一种技术方案如下:一种表达载体,所述表达载体可表达所述的纳米抗体或含有所述的基因。
进一步,表达载体为噬菌体或质粒。
本发明解决上述技术问题的另一种技术方案如下:一种宿主细胞,所述宿主细胞含有所述的表达载体。
进一步,所述宿主细胞为大肠杆菌。
本发明解决上述技术问题的另一种技术方案如下:一种所述具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体的制备方法,通过噬菌体展示技术筛选到所述的纳米抗体,包括以下步骤:
(1)制备去掉其GPI区域且羧基端带有FLAG标签的GPC3ΔGPI-FLAG重组蛋白;
(2)将纳米抗体噬菌体文库与GPC3ΔGPI-FLAG重组蛋白孵育,将结合GPC3ΔGPI-FLAG重组蛋白的噬菌体洗脱并侵染宿主细胞,利用其产生的噬菌体再与GPC3ΔGPI-FLAG重组蛋白重新孵育,进行新一轮筛选,重复筛选三次;
(3)从筛选后最终得到的含噬菌体质粒的宿主细胞中挑取单克隆进行测序,将三个互补决定区CDR1、CDR2、CDR3序列相同的克隆视为同一克隆株,根据测序结果,选取重复率高的克隆株;
(4)通过PCR扩增、限制性内切酶酶切和T4连接酶连接将所选取的克隆中的纳米抗体基因片段连接至表达载体中,将表达载体转化至蛋白原核表达菌株,接种至培养基进行培养后,收集细菌并进行周质蛋白提取、纯化,得到所述GPC3的纳米抗体。
本发明解决上述技术问题的另一种技术方案如下:一种所述具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体的应用,其可用于GPC3的功能研究或肝细胞癌诊断试剂、治疗药物的开发,包括GPC3蛋白亚细胞定位的分析试剂、GPC3蛋白水平的检测试剂、肝细胞癌分子成像诊断试剂、药物靶向输送试剂、嵌合抗原受体T细胞免疫疗法、纳米抗体药物的开发。
本发明的有益效果是:本发明通过噬菌体展示技术筛选出GPC3的纳米抗体,该纳米抗体特异性结合定位于细胞膜上的GPC3,且抗体稳定性高。本发明利用原核表达来制备纳米抗体,操作简单、成本低廉。作为肝细胞癌的靶向分子,该纳米抗体可用于肝细胞癌分子成像诊断、药物靶向输送、嵌合抗原受体T细胞免疫治疗或纳米抗体药物的开发,该纳米抗体亦可用于GPC3的功能研究。
附图说明
图1为表达载体pLenti6/V5-GPC3ΔGPI-FLAG的构建流程图。
图2为利用FLAG M2磁珠对稳定细胞株培养液中的分泌蛋白GPC3ΔGPI-FLAG进行纯化,并利用FLAG抗体进行免疫印迹检测确认目的蛋白的结果。
图3为纳米抗体表达载体pET22b-Nb-His的构建流程图。
图4为运用GPC3免疫共沉淀纳米抗体方法检测抗原-抗体结合的结果。
图5为运用纳米抗体免疫共沉淀GPC3方法检测抗原-抗体结合的结果。
图6为免疫荧光染色实验检测纳米抗体与GPC3共定位的结果。
图7为免疫荧光染色实验检测纳米抗体与肝癌细胞特异性结合的结果。
图8为酶联免疫吸附实验检测所筛选的纳米抗体与已知GPC3纳米抗体HN3及GPC3单克隆抗体GPC3-mAb的稳定性实验结果。
具体实施方式
以下对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
实施例1GPC3重组蛋白的制备
制备羧基端带有FLAG标签的GPC3重组蛋白。
(1)根据GPC3在NCBI中的编码基因序列(NCBI Reference Sequence:NM_004484.4),并以Uni Prot中GPC3的氨基酸序列(Uni Prot Reference Sequence:P51654-1)为准进行F359S突变,去掉其GPI区域(1690-1740bp),将缺失GPI位点的GPC3ΔGPI与FLAG标签融合的序列进行人工合成(深圳华大基因科技有限公司),5’端和3’端分别带有EcoRI和NotI酶切位点。GPC3ΔGPI编码的氨基酸序列如SEQ ID NO:9所示(Met Ala Gly Thr ValArg Thr Ala Cys Leu Val Val Ala Met Leu Leu Ser Leu Asp Phe Pro Gly Gln AlaGln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser Phe Phe GlnArg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp LeuGln Val Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys TyrGln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu LeuLys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val ValArg His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser Leu ThrPro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile LeuGly Ser Asp Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe ProVal Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile AsnGlu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys LeuIle Met Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala LeuAsn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp CysGly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met ValLys Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly Val ValGlu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn GlyMet Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile HisAsp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly LysLeu Cys Ala His Ser G n Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp LeuPhe Ile Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu SerSer Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser AlaLeu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys TrpAsn Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly Met LysAsn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser GlnIle Il e Asp Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro LysGly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys GlyAsp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys AsnGln Leu Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro GlyAsn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu GlyAsn Val His Ser Pro Leu Lys),其核苷酸序列如SEQ ID NO:10所示(atggccgggaccgtgcgcac cgcgtgcttg gtggtggcga tgctgctcag cttggacttc ccgggacagg cgcagcccccgccgccgccg ccggacgcca cctgtcacca agtccgctcc ttcttccaga gactgcagcc cggactcaagtgggtgccag aaactcccgt gccaggatca gatttgcaag tatgtctccc taagggccca acatgctgctcaagaaagat ggaagaaaaa taccaactaa cagcacgatt gaacatggaa cagctgcttc agtctgcaagtatggagctc aagttcttaa ttattcagaa tgctgcggtt ttccaagagg cctttgaaat tgttgttcgccatgccaaga actacaccaa tgccatgttc aagaacaact acccaagcct gactccacaa gcttttgagtttgtgggtga atttttcaca gatgtgtctc tctacatctt gggttctgac atcaatgtag atgacatggtcaatgaattg tttgacagcc tgtttccagt catctatacc cagctaatga acccaggcct gcctgattcagccttggaca tcaatgagtg cctccgagga gcaagacgtg acctgaaagt atttgggaat ttccccaagcttattatgac ccaggtttcc aagtcactgc aagtcactag gatcttcctt caggctctga atcttggaattgaagtgatc aacacaactg atcacctgaa gttcagtaag gactgtggcc gaatgctcac cagaatgtggtactgctctt actgccaggg actgatgatg gttaaaccct gtggcggtta ctgcaatgtg gtcatgcaaggctgtatggc aggtgtggtg gagattgaca agtactggag agaatacatt ctgtcccttg aagaacttgtgaatggcatg tacagaatct atgacatgga gaacgtactg cttggtctct tttcaacaat ccatgattctatccagtatg tccagaagaa tgcaggaaag ctgaccacca ctattggcaa gttatgtgcc cattctcaacaacgccaata tagatccgct tattatcctg aagatctctt tattgacaag aaagtattaa aagttgctcatgtagaacat gaagaaacct tatccagccg aagaagggaa ctaattcaga agttgaagtc tttcatcagcttctatagtg ctttgcctgg ctacatctgc agccatagcc ctgtggcgga aaacgacacc ctttgctggaatggacaaga actcgtggag agatacagcc aaaaggcagc aaggaatgga atgaaaaacc agttcaatctccatgagctg aaaatgaagg gccctgagcc agtggtcagt caaattattg acaaactgaa gcacattaaccagctcctga gaaccatgtc tatgcccaaa ggtagagttc tggataaaaa cctggatgag gaagggtttgaaagtggaga ctgcggtgat gatgaagatg agtgcattgg aggctctggt gatggaatga taaaagtgaagaatcagctc cgcttccttg cagaactggc ctatgatctg gatgtggatg atgcgcctgg aaacagtcagcaggcaactc cgaaggacaa cgagataagc acctttcaca acctcgggaa cgttcattcc ccgctgaag)。(2)利用限制性内切酶EcoRI和Not I(New England Biolabs公司)对GPC3ΔGPI-FLAG DNA片段和经改造多克隆位点两端含有SfiI酶切位点的pcDNA3.1载体(中间载体)进行双酶切,对所获得的片段利用T4连接酶(Thermo Fisher Scientific公司)进行连接反应,测序验证。经SfiI酶切将GPC3ΔGPI-FLAG连接至慢病毒包装载体pLenti6/V5中,最终获得正确的pLenti6/V5-GPC3ΔGPI-FLAG载体(见图1)。(3)将pLenti6/V5-GPC3ΔGPI-FLAG以及慢病毒包装载体pLP1、pLP2和pVSVG共转染至HEK293FT细胞中,从上清中获得相应慢病毒。将慢病毒转导至HEK293T细胞,经抗生素筛选获得稳定表达GPC3ΔGPI-FLAG重组蛋白的细胞株。将细胞株于37℃、5%CO2条件下培养2天,收集培养上清液,按照FLAG M2磁珠(Sigma-Aldrich公司)说明书对GPC3ΔGPI-FLAG重组蛋白进行纯化。
实施例2针对GPC3的纳米抗体噬菌体文库筛选
(1)包被抗原:按FLAG M2磁珠说明书,对分泌至培养上清液中的GPC3ΔGPI-FLAG蛋白进行纯化,制备GPC3ΔGPI-FLAG蛋白包被的磁珠,并通过考马斯亮蓝染色及免疫印迹检测进行验证。如图2所示,由左图考马斯亮蓝染色结果可知纯化得到的蛋白大小位于72-85kD之间,条带单一,表明纯度较高。右图为利用GPC3ΔGPI-FLAG重组蛋白的FLAG标签抗体进行免疫印迹检测结果,显示蛋白大小与考马斯亮蓝染色结果一致,表明纯化得到的蛋白为GPC3ΔGPI-FLAG目的蛋白。GPC3蛋白具有数个糖基化位点,拖尾弥散条带应为糖基化所致。箭头处指示为GPC3ΔGPI-FLAG重组蛋白目的条带位置。(2)纳米抗体库的预处理:取1ml含5%(g/ml)BSA及0.1%Tween-20的TBS缓冲溶液(pH 7.4),加入本实验室构建的纳米抗体噬菌体展示文库,使溶液中噬菌体达到约1×1011pfu.。混匀后加入20μl FLAG M2磁珠,室温旋转孵育60min。(3)含有纳米抗体的噬菌体与GPC3的结合:将上述预处理后的纳米抗体噬菌体溶液转移至新的离心管中,加入20μl包被有GPC3ΔGPI-FLAG的磁珠(GPC3ΔGPI-FLAG蛋白量约2μg),室温旋转孵育60min。(4)洗涤:弃去上述纳米抗体噬菌体溶液,用含有TBST(0.1%Tween-20)溶液洗涤磁珠3次。(5)洗脱:向含有磁珠的离心管中加入100μl 0.1M三乙胺溶液,常温温和振荡10min,然后加入100μl 1M Tris-HCl(pH 6.8),振荡混匀,室温静置5min。(6)侵染:将洗脱液加入1ml大肠杆菌SS320培养液(OD600值为0.6),37℃孵育40min。(7)计数:将SS320培养液离心5000rpm,4min,弃上清,加入200μl LB混匀,按一定稀释度取部分菌液涂于计数平板,过夜培养。(8)计数完毕后用1ml ddH2O将平板上的菌进行收集,并取一定量菌液于2×YT(含2%Glucose)中,37℃,220rpm培养15min。(9)加入辅助噬菌体M13KO7(New England Biolabs公司),侵染SS320,37℃孵育60min,制备并纯化噬菌体用于下一轮筛选。(10)将收集的噬菌体纳米抗体文库按照实施例2的步骤再进行下一轮的筛选,共筛选三次。
实施例3纳米抗体的制备
实施例2中,完成第三轮筛选噬菌体侵染后,将大肠杆菌SS320涂布平板,挑取含噬菌体质粒的单克隆进行测序。利用Vector NTI软件分析各个抗体克隆的基因序列并进行比对,将互补区CDR1、CDR2、CDR3序列相同的克隆视为同一克隆株。根据测序结果,选取其中一个高重复率的克隆株,标记为G11克隆,所示DNA序列如SEQ ID NO:8所示,其编码氨基酸序列如SEQ ID NO:7所示。通过PCR扩增、限制性内切酶酶切和T4连接酶连接将所选G11纳米抗体的核苷酸片段连接至表达载体pET22b中,具体过程如下:(1)依据上述测序结果设计用于扩增纳米抗体的核苷酸片段的PCR引物:上游引物catgactagt caagttcaat tagtc(SEQ IDNO:11),下游引物cgcggatcca agcttgaatt c(SEQ ID NO:12)。(2)以G11噬菌体质粒为模板,PCR扩增纳米抗体的DNA片段,然后通过SpeI和EcoRI限制性内切酶(New EnglandBiolabs公司)酶切,用T4连接酶(Thermo Fisher Scientific公司)连接至表达载体pET22b中,经过DNA序列测定,获得含有编码G11核苷酸序列的重组质粒pET22b-G11-His(见图3)。(3)随机挑取一个未经任何筛选的纳米抗体噬菌体基因库侵染大肠杆菌SS320后的单克隆菌株,将其所表达的纳米抗体作为阴性对照抗体(Negative control,NC),并依据实施例3(1)和实施例3(2)构建对照纳米抗体表达载体pET22b-NC-His(见图3)。(4)将重组质粒pET22b-G11-His和pET22b-NC-His分别转化至大肠杆菌BL21,获得表达融合His标签的纳米抗体的工程菌,并分别接种至LB培养基中,37℃,220rpm培养至培养液OD600值为0.6,加入IPTG至终浓度为1mM,然后在18℃,220rpm诱导表达12h。培养结束后,收集大肠杆菌并进行周质蛋白提取,按照His标签蛋白纯化磁珠(苏州海狸生物医学工程有限公司)使用说明书,纯化携带了His标签的纳米抗体。
实施例4GPC3免疫共沉淀纳米抗体实验
(1)依据实施例1获得结合GPC3ΔGPI-FLAG的磁珠。(2)依据实施例3中获得含Nb-His的细胞裂解液,利用10×Buffer W(1M Tris/HCl,pH 8.0,1.5M NaCl,10mM EDTA)将细胞裂解液稀释成1×Buffer W溶液体系,取500μl加入5μl结合GPC3ΔGPI-FLAG的磁珠,4℃旋转孵育2h。(3)Buffer W洗涤磁珠3次,弃去洗涤溶液,每管加入10μl 1×SDS凝胶加样缓冲液,振荡混匀,煮沸10min。(4)利用蛋白免疫印迹(Western blot,WB)检测免疫共沉淀后样品中GPC3和纳米抗体。
检测结果如图4所示,其中NC为阴性对照纳米抗体,左侧Input为施加的纳米抗体蛋白;右侧为免疫沉淀的纳米抗体,用未结合抗原的空白FLAG M2磁珠(Blank Beads)作为对照组,以排除纳米抗体与磁珠的直接结合。免疫印迹检测中用anti-His抗体检测纳米抗体,条带强弱代表纳米抗体蛋白量的多少。可以看出:阴性对照纳米抗体NC、G11均不与磁珠结合,而与包被了GPC3ΔGPI-FLAG磁珠结合的G11的蛋白条带较强,表明G11纳米抗体能高特异性结合GPC3。
实施例5G11纳米抗体免疫共沉淀GPC3实验
(1)依据实施例3获得成功结合了Nb-His的磁珠。(2)加入5ml实施例1中所得的含GPC3ΔGPI-FLAG分泌蛋白的培养液,4℃旋转孵育2h。(3)弃去培养液,TBST洗涤磁珠3次。弃去洗涤溶液,每管加入10μl SDS凝胶加样缓冲液,振荡混匀,煮沸10min。(4)利用蛋白免疫印迹检测免疫共沉淀后样品中的GPC3和纳米抗体。
检测结果如图5所示,其中NC为阴性对照纳米抗体,上图为Input,表示该实验各组中加入的GPC3ΔGPI-FLAG重组蛋白;中图表示用于实验检测的各组磁珠上结合的纳米抗体量;下图为免疫沉淀的GPC3ΔGPI-FLAG重组蛋白。结果显示:阴性对照纳米抗体NC不与GPC3ΔGPI-FLAG结合,而G11组能高效沉淀GPC3ΔGPI-FLAG,表明G11高特异性地结合GPC3。
实施例6纳米抗体识别细胞膜表面GPC3的免疫荧光染色实验
(1)为进一步确认纳米抗体G11能识别并结合定位于细胞膜的GPC3,我们将mCherry融合于GPC3的N端,根据实施例1的方法构建相关载体,通过慢病毒包装、转导,获得稳定表达mCherry-GPC3的HEK293T细胞株。(2)将传代培养的该细胞株经胰酶充分消化后获得单细胞悬液,接种至细胞爬片。(3)将细胞爬片用37℃的PBS(pH7.4,下同)漂洗,用4%PFA(多聚甲醛)于37℃固定10min,PBS漂洗。(4)在2%BSA/TBS中室温下封闭30min。(5)利用2%BSA/TBS稀释纳米抗体至10μg/mL,4℃孵育过夜。(6)TBST漂洗,加入2%BSA/TBS稀释的anti-HA(Cell signaling公司),37℃孵育2h。(7)TBST漂洗,加入2%BSA/TBS稀释的偶联Alexa Fluor 488荧光基团的二抗(Thermo Fisher Scientific公司),37℃孵育1h。(8)TBST漂洗,用5μg/mL DAPI孵育2min对细胞核进行染色。制片后在荧光共聚焦显微镜下观察、拍照。
检测结果如图6A所示,mCherry荧光信号分布表明mCherry-GPC3定位于细胞膜及细胞质中,而指示纳米抗体G11定位的Alexa Fluor 488荧光信号仅出现于细胞膜上。由于本实施例中未施加表面活性剂处理,细胞膜结构未被破坏,因此G11仅结合细胞膜定位的GPC3蛋白。对直线线条区域分析结果如图6B所示,横坐标为直线线条上距离左端点的距离,纵坐标表示荧光信号强度,可见mCherry和Alexa Fluor 488两种荧光信号的强度随位置变动同步变化,而且矩形区域的计算结果显示(图6C),两种荧光的皮尔森相关系数为0.929。以上结果表明,纳米抗体G11和细胞膜定位的mCherry-GPC3具有良好的共定位,说明该抗体能特异性地识别并结合细胞膜上的GPC3蛋白。
实施例7纳米抗体识别肝癌细胞的免疫荧光染色实验
为进一步验证纳米抗体G11是否能特异性识别GPC3阳性的肝癌细胞,我们根据实施例6的方法,利用该纳米抗体对高表达GPC3的人肝细胞癌细胞系HepG2进行免疫荧光染色。不表达GPC3的人表皮鳞癌细胞系A431作为阴性对照细胞系。
检测结果如图7所示,阴性对照细胞A431细胞膜上没有检测到纳米抗体G11的荧光信号,但HepG2细胞膜上可检测到较强的荧光信号,表明G11能特异性地识别、结合肝细胞癌细胞。
实施例8纳米抗体的稳定性测试实验
通过酶联免疫吸附实验检测、比较纳米抗体G11、已知GPC3纳米抗体HN3和GPC3单克隆抗体GPC3-mAb的热稳定性。
(1)按实施例1表达并纯化抗原蛋白GPC3ΔGPI-FLAG。(2)依据实施例3表达、纯化纳米抗体G11。HN3纳米抗体从Creative Biolabs购置,是HN3-hFc融合抗体[Feng M,Gao W,Wang R,et al.2013.Therapeutically targeting giypican-3via a conformation-specific single-domain antibodyin hepatocellular carcinoma.PNAS 110(12):E1083-E1091]。GPC3-mAb从成都正能生物技术有限责任公司购置。(3)分别测定G11、HN3-hFc和GPC3-mAb抗体浓度,取2μg置于1×PBS(pH7.4)溶液环境中,分别于4℃、20℃、37℃、55℃、70℃及90℃环境下处理2h。(4)将等量的GPC3ΔGPI-FLAG蛋白稀释于包被液中,加入微孔板,4℃包被过夜。(5)用Blocking buffer(PBS,0.05%Tween-20,1%BSA)封闭后,将上述经不同温度处理的G11、HN3-hFc和GPC3-mAb抗体加入微孔板,37℃孵育1小时。(6)清洗后,分别向G11微孔和HN3-hFc微孔加入小鼠His标签抗体和小鼠hFc抗体,37℃孵育1小时。GPC3-mAb微孔略去此步。(7)清洗后,加入新鲜稀释的HRP偶联酶标二抗(Thermo FisherScientific公司,1:10000稀释),37℃孵育30min。(8)加入TMB显色液(北京索莱宝科技有限公司),37℃孵育20min显色。(9)显色后向各反应孔中加入显色终止液(北京索莱宝科技有限公司),利用酶标仪检测各反应孔450nm的光吸收值。
检测结果如图8所示,同等条件下,小鼠单克隆抗体GPC3-mAb的抗原结合能力随温度升高急剧下降,在70℃时抗原结合能力下降95.5%(以4℃时450nm的光吸收值为参照,下同);文献报道的纳米抗体HN3的热稳定性高于GPC3-mAb,但依然随温度升高稳定性明显下降,在70℃时抗原结合能力下降42.9%,90℃时下降58.3%;而本发明中的纳米抗体G11的光吸收值随温度升高变化不大,在70℃条件下抗原结合能力仅下降16.7%,而在90℃时下降30.8%,表明G11的热稳定性高于HN3及GPC3-mAb。纳米抗体骨架区维持纳米抗体的基本结构,影响其蛋白稳定性,本发明筛选得到的纳米抗体G11的骨架区FR1-4的氨基酸序列分别如SEQ ID NO:13-16所示。
上述实施例为本发明的优选实施方式,并不对本发明构成任何限制,其他任何在未背离本发明的精神实质与原理下所作的替代、简化、组合等改变或修饰,均包含在本发明的保护范围之内。
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Claims (10)

1.一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体,其特征在于,所述纳米抗体包括3个互补决定区,分别为CDR1、CDR2和CDR3;其中,CDR1的氨基酸序列如SEQ IDNO.1所示,CDR2的氨基酸序列由SEQ ID NO.2所示,CDR3的氨基酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体,其特征在于,所述纳米抗体还包括与三个互补决定区交替连接的四个骨架区,分别为FR1、FR2、FR3和FR4;其中,FR1的氨基酸序列如SEQ ID NO.13所示,FR2的氨基酸序列由SEQ IDNO.14所示,FR3的氨基酸序列如SEQ ID NO.15所示,FR4的氨基酸序列如SEQ ID NO.16所示。
3.根据权利要求2所述一种具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体,其特征在于,所述纳米抗体的氨基酸序列如SEQ ID NO:7所示。
4.一种编码如权利要求1至3任一项所述具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体的基因,其特征在于,包括三段分别编码其3个互补决定区的核苷酸序列,其中,编码CDR1的核苷酸序列如SEQ ID NO.4所示,编码CDR2的核苷酸序列如SEQ ID NO.5所示,编码CDR3的核苷酸序列如SEQ ID NO.6所示。
5.根据权利要求4所述一种编码具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO:8所示。
6.一种表达载体,其特征在于,所述表达载体可表达权利要求1至3任一项所述的纳米抗体或含有权利要求4或5所述的基因。
7.一种宿主细胞,其特征在于,所述宿主细胞含有如权利要求6所述的表达载体。
8.根据权利要求7所述一种宿主细胞,其特征在于,所述宿主细胞为大肠杆菌。
9.一种如权利要求1至3任一项所述具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体的制备方法,其特征在于,通过噬菌体展示技术筛选到所述的纳米抗体,包括以下步骤:
(1)制备去掉其GPI区域且羧基端带有FLAG标签的GPC3ΔGPI-FLAG重组蛋白;
(2)将纳米抗体噬菌体文库与GPC3ΔGPI-FLAG重组蛋白孵育,将结合GPC3ΔGPI-FLAG重组蛋白的噬菌体洗脱并侵染宿主细胞,利用其产生的噬菌体再与GPC3ΔGPI-FLAG重组蛋白重新孵育,进行新一轮筛选,重复筛选三次;
(3)从筛选后最终得到的含噬菌体质粒的宿主细胞中挑取单克隆进行测序,将三个互补决定区CDR1、CDR2、CDR3序列相同的克隆视为同一克隆株,根据测序结果,选取重复率高的克隆株;
(4)通过PCR扩增、限制性内切酶酶切和T4连接酶连接将所选取的克隆中的纳米抗体基因片段连接至表达载体中,将表达载体转化至蛋白原核表达菌株,接种至培养基进行培养后,收集细菌并进行周质蛋白提取、纯化,得到所述磷脂酰肌醇蛋白聚糖3的纳米抗体。
10.一种如权利要求1至3任一项所述具有突出高稳定性的磷脂酰肌醇蛋白聚糖3的纳米抗体的应用,其特征在于,其可用于磷脂酰肌醇蛋白聚糖3的功能研究或肝细胞癌诊断试剂、治疗药物的开发,包括磷脂酰肌醇蛋白聚糖3蛋白亚细胞定位的分析试剂、磷脂酰肌醇蛋白聚糖3蛋白水平的检测试剂、肝细胞癌分子成像诊断试剂、药物靶向输送试剂、嵌合抗原受体T细胞免疫疗法、纳米抗体药物的开发。
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