CN114656568A - 一种ceacam5靶向的近红外荧光纳米抗体探针及其制备方法和应用 - Google Patents
一种ceacam5靶向的近红外荧光纳米抗体探针及其制备方法和应用 Download PDFInfo
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- CN114656568A CN114656568A CN202210572690.2A CN202210572690A CN114656568A CN 114656568 A CN114656568 A CN 114656568A CN 202210572690 A CN202210572690 A CN 202210572690A CN 114656568 A CN114656568 A CN 114656568A
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Abstract
本发明公开一种CEACAM5靶向的近红外荧光纳米抗体探针及其制备方法和应用，涉及分子影像领域，该探针由CEACAM5纳米抗体重组蛋白和近红外荧光染料两部分组成。本发明实现了对CEACAM5分子的可视化，进一步实现了对CEACAM5分子阳性肿瘤的诊断。所述探针具有制备工艺简单，成本低廉，特异性高，稳定性高，且易于临床转化等优点。
Description
技术领域
本发明涉及分子影像领域，尤其涉及CEACAM5靶向的近红外荧光纳米抗体探针及其制备方法和应用。
背景技术
1993年比利时科学家Hamers-Casterman等在《Nature》杂志上首次报道了在骆驼血清中大量存在的一种天然缺失轻链的抗体，即重链抗体(Heavy-chain antibodies,HCAbs)。单域重链抗体是指仅由重链抗体可变区组成的基因工程抗体，又称为VHH抗体(Variable Domain of Heavy Chain of Heavy Chain Antibody)或者纳米抗体(Nanobody, NB)。与普通抗体相比，骆驼重链抗体除了缺少轻链之外，其重链可变区与铰链区之间没有CH1区。恒定区CH1是与轻链锚定的部位，在纳米抗体的基因组中存在，但在mRNA形成中被剪掉，纳米抗体靠仅有的3个CDRs (complementarity determining region,CDR)就具备抗原的特异性和高亲和力，而普通抗体则需要6 CDRs，是目前已知的可结合目标抗原的最小单位。和普通抗体相比，纳米抗体分子量小，结构简单，易于进行基因改造，体积小，抗原特异性好，组织穿透力强，稳定性高，在疾病的诊断及治疗方面有广阔的应用前景。
癌胚抗原相关细胞粘附分子 5 (CEACAM5) 也称癌胚抗原 (CEA)，在正常组织中限制性表达，而在结直肠癌、胰腺癌、乳腺癌、胃癌以及小细胞肺癌等中过表达。因此CEACAM5可作为肿瘤标志物用于肿瘤的诊断和治疗。
近红外荧光染料IRDye800具备成像背景低，灵敏度高，信号稳定等特点，适用于活体成像以及术中导航。本发明将将具备CEACAM5高亲和力的纳米抗体与近红外荧光染料IRDye800CW偶联，制备出一种用于诊断CEACAM5分子阳性肿瘤的近红外荧光纳米抗体探针。该探针能够在体内外靶向识别高表达CEACAM5分子阳性肿瘤细胞和瘤灶，在荧光成像和基于荧光成像的手术导航中有良好的应用前景。
发明内容
本发明的目的在于提供用于活体内荧光成像和基于荧光成像的手术导航的近红外荧光纳米抗体探针。
本发明的目的可以通过以下技术方案实现：
为实现上述目的，本发明提供了一种新型的针对CEACAM5分子的纳米抗体重组蛋白，NB41-ABD，该纳米抗体重组蛋白由纳米抗体NB41经羧基端修饰获得，所述羧基端修饰为：在纳米抗体NB41的羧基端修饰白蛋白结合域(Albumin-Binding Domain, ABD)序列。该纳米抗体重组蛋白的氨基酸序列为：QLQLVESGGGLVQAGGSLRLSCAASGSLFRINAMAWFRQAPGKQRELVAAITSAGSTNYADFVKGRFTISADNAKNTLYLQMNSLKPEDTAVYYCNTPWPVGRDYWGQGTQVTVSSEPKTPKPQPAAAQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPAAAGAAA (SEQID NO:2)
上述纳米抗体重组蛋白NB41-ABD的核苷酸序列为：cagttgcagctcgtggagtctggtggaggcttggtgcaggctggggggtccctgagactctcctgtgcagcctctggaagcctcttcaggatcaatgccatggcctggttccgccaggctccagggaagcagcgcgagttggtcgcagctattactagtgctggtagtacaaactatgcagatttcgtgaagggccgattcaccatctccgcagacaacgccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacacagccgtctattactgtaatacaccctggcccgtagggagggactactggggccaggggacccaggtcaccgtctcctcagaacccaagacaccaaaaccacaaccagcagcagctcaacatgatgaagctgttgatgcgaactccctggcagaagcgaaagttctggccaaccgtgaactggataaatacggcgtgtccgactactacaaaaacctgatcaacaacgcaaaaactgttgaaggcgttaaggcactgatcgatgaaattctggcagcgctgccagcagctgctggcgctgctgct (SEQ ID NO:4)。
本发明还提供了一种新型的针对CEACAM5分子的纳米抗体重组蛋白，NB41-ABD-C，该纳米抗体重组蛋白的氨基酸序列为：
QLQLVESGGGLVQAGGSLRLSCAASGSLFRINAMAWFRQAPGKQRELVAAITSAGSTNYADFVKGRFTISADNAKNTLYLQMNSLKPEDTAVYYCNTPWPVGRDYWGQGTQVTVSSEPKTPKPQPAAAQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPAAACAAA (SEQ ID NO:3)
上述纳米抗体重组蛋白NB41-ABD-C的核苷酸序列为：
cagttgcagctcgtggagtctggtggaggcttggtgcaggctggggggtccctgagactctcctgtgcagcctctggaagcctcttcaggatcaatgccatggcctggttccgccaggctccagggaagcagcgcgagttggtcgcagctattactagtgctggtagtacaaactatgcagatttcgtgaagggccgattcaccatctccgcagacaacgccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacacagccgtctattactgtaatacaccctggcccgtagggagggactactggggccaggggacccaggtcaccgtctcctcagaacccaagacaccaaaaccacaaccagcagcagctcaacatgatgaagctgttgatgcgaactccctggcagaagcgaaagttctggccaaccgtgaactggataaatacggcgtgtccgactactacaaaaacctgatcaacaacgcaaaaactgttgaaggcgttaaggcactgatcgatgaaattctggcagcgctgccagcagctgcttgtgctgctgct (SEQ ID NO:5)。
本发明所述的近红外荧光纳米抗体探针通过特异性靶向CEACAM5至肿瘤部位，并且在肿瘤部位有很好的聚集和滞留，具有较高的肿瘤背景比，适合用作活体内荧光成像和基于荧光成像的手术导航的探针。
本发明是一种高效的肿瘤标志物CEACAM5的检测技术，首先外源表达识别CEACAM5的纳米抗体重组蛋白NB41，NB41-ABD或NB41-ABD-C。然后通过标记近红外荧光染料形成近红外荧光纳米抗体探针。
所述近红外荧光染料优选IRDye800CW、IRDye680RD、Cy7.5、Cy5.5中的任意一个。
本发明还提供一种增强近红外荧光纳米抗体探针体内循环时间从而增强信号的策略，通过在纳米抗体重组蛋白NB41的羧基端修饰白蛋白结合域(Albumin-BindingDomain, ABD)序列，所形成的纳米抗体重组蛋白NB41-ABD和NB41-ABD-C，能有效降低红外荧光纳米抗体探针体在肾脏的累积，提高其体内循环时间，增强肿瘤背景比。
本发明还提供一种近红外荧光纳米抗体探针NB41-ABD-IR800，并报道其在诊断CEACAM5阳性肿瘤中的应用。本发明通过创制CEACAM5特异性近红外荧光纳米抗体探针NB41-ABD-IR800，实现了对CEACAM5分子表达的无创可视化，进一步实现了CEACAM5阳性肿瘤的无创诊断，具有制备工艺简单，成本低廉，特异性高，稳定性高，且易于临床转化等优点。
有益效果：
本发明提供的用于检测肿瘤标志物CEACAM5的基于纳米抗体重组蛋白的近红外荧光探针、制备方法及应用，与现有技术相比，具有以下优势：能有效降低红外荧光纳米抗体探针体在肾脏的累积，减轻肾脏负荷，同时提高探针在体内循环时间，从而有效增强肿瘤背景比。本发明所述的CEACAM5特异性近红外荧光纳米抗体探针NB41-ABD-IR800，实现了对CEACAM5表达的无创可视化，进一步实现了对CEACAM5分子阳性肿瘤的无创诊断，具有制备工艺简单，成本低廉，特异性高，稳定性高，且易于临床转化等优点。
附图说明
图1为NB41-ABD的SDS-PAGE检测结果。
图2为NB41-ABD-IR800的ELISA结果。
图3为NB41-ABD-IR800的流式细胞术检测结果。
图4为NB41-ABD-IR800在小鼠皮下结肠癌模型中的荧光成像。
图5为NB41-ABD-IR800在小鼠皮下结肠癌模型中的荧光成像的定量分析结果。
具体实施方式
以下结合说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于本文中所述的特定方法、方案、细胞系、构筑体和试剂，并且同样可改变。除非另有定义，本说明书所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是用于限制本发明。
实施例1
本实施例中，纳米抗体重组蛋白NB41-ABD和NB41-ABD-C序列中能够识别CEACAM5的部分蛋白质氨基酸序列来源于专利CN 112028997 B (原始氨基酸序列为该专利中的SEQID NO:3)，氨基酸序列如SEQ ID NO:1所示。
对SEQ ID NO:1所示序列的羧基末端修饰上白蛋白结合域后得到本发明中具备额外新功能的能识别CEACAM5的纳米抗体重组蛋白NB41-ABD (SEQ ID NO:2)和NB41-ABD-C(SEQ ID NO:3)。
具体实施方法如下：如NB41-ABD或NB41-ABD-C，首先根据重组蛋白质序列按照大肠杆菌核苷酸密码子反推得到识别CEACAM5的纳米抗体重组蛋白NB41-ABD(SEQ ID NO:4)或NB41-ABD-C(SEQ ID NO:5)的核苷酸序列，合成核苷酸序列 (SEQ ID NO:4)并克隆插入表达载体pET28a质粒中，并将该质粒转入大肠杆菌E .coli BL21(DE3)。该菌株即为生产能识别CEACAM5的纳米抗体重组蛋白NB41-ABD或NB41-ABD-C的发酵菌株E .coli BL21(DE3)-NB41-ABD或E .coli BL21(DE3)- NB41-ABD-C。
实施例2
诱导表达纳米抗体重组蛋白NB41-ABD。
具体实施方式如下：将贮存于甘油的重组BL21(DE3)接种于含卡那霉素(50 mg/L)的LB培养基，在37℃培养，当OD600达到0.6左右时，加入IPTG（0.1mM）在16℃诱导纳米抗体表达，诱导时长为16小时，通过离心收集菌体；菌体重悬后进行高压破碎；离心后吸取上清液。上清液经0 .22μm滤膜过滤后，经镍柱进行初步纯化；随后经分子筛进行进一步纯化，储存于保存缓冲液中；通过nanodrop测定蛋白浓度，并用SDS-PAGE进一步确认。如图1所示，所表达的纳米抗体重组蛋白纯度均大于90%。
实施例3
CEACAM5特异性近红外荧光纳米抗体探针NB41-ABD-IR800的制备。
具体实施方式如下：取500 mg纳米抗体重组蛋白NB41-ABD，加入IRDye800CW近红外荧光染料（蛋白与染料的摩尔比为2-4），反应体系溶剂为PBS，体积为500 mL。将该反应混合体系室温下避光，置于摇床上反应2h。然后通过PD10层析柱进行分离。所得产物通过nanodrop测定A280nm和A780nm处的吸光值，计算后得到产物浓度。所得产物避光置于4℃保存。
实施例4
ELISA检测NB41-ABD-IR800对CEACAM5的亲和力。
具体实施方式如下：抗原包被，人CEACAM5重组蛋白200 ng/孔，每孔100uL，4℃冰箱孵育过夜；次日吸走未结合的蛋白，wash buffer轻柔洗两次。37℃以含1%BSA的PBS封闭1h；加入以浓度梯度稀释的NB41-ABD-IR800，37℃孵育1h；wash buffer洗3遍去除未结合的抗体；加入anti-his-HRP二抗在37℃孵育1小时；wash buffer洗3-5遍去除未结合的抗体；加入TMB底物，37℃， 15mins 避光孵育；每孔50ul的终止液，15mins内检测；酶标仪测量信号。结果如图2所示，NB41-ABD-IR800保持了对CEACAM5的高亲和力。
实施例5
流式细胞术检测NB41-ABD-IR800对结肠癌细胞系的结合力。
具体实施方式如下：使用Acutase消化液消化细胞(人结肠腺癌细胞系LS174T)，200µLPBS重悬；加入1µM NB41-ABD-IR800，冰上或4℃孵育30mins；PBS清洗3遍，加入anti-his-FITC二抗冰上避光孵育30mins；PBS清洗后重悬至流式管，上机检测。结果如图3所示，NB41-ABD-IR800对人结肠腺癌细胞系LS174T (CEACAM5阳性)有较强亲和力。
实施例6
NB41-ABD-IR800在小鼠皮下结肠癌模型中的荧光成像。
将荷瘤小鼠分成两组，分别通过尾静脉注射等量NB41-IR800和NB41-ABD-IR800，应用IVIS小动物活体成像系统进行持续检测。NB41-ABD-IR800在注射0.5h后便能在肿瘤处聚集，8h后达到较强的肿瘤背景比，并持续增长 (图4，5)。证明所构建的近红外荧光探针NB41-ABD-IR800可以特异性的识别高表达CEACAM5分子的肿瘤。
需要说明的是，本发明的说明书及其附图中给出了本发明的较佳实施例，但是，本发明可以通过许多不同的形式来实现，并不限于本说明书所描述的实施例，这些实施例不作为对本发明内容的额外限制，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。并且，上述各技术特征继续相互组合，形成未在上面列举的各种实施例，均视为本发明说明书记载的范围；进一步地，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
序列表
<110> 中山大学附属第五医院
<120> 一种CEACAM5靶向的近红外荧光纳米抗体探针及其制备方法和应用
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<221> CONFLICT
<222> (1)..(191)
<223> NB41-ABD氨基酸序列
<400> 2
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Leu Phe Arg Ile Asn
20 25 30
Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Ser Ala Gly Ser Thr Asn Tyr Ala Asp Phe Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Thr Pro Trp Pro Val Gly Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala
115 120 125
Gln His Asp Glu Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys Val
130 135 140
Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys
145 150 155 160
Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile
165 170 175
Asp Glu Ile Leu Ala Ala Leu Pro Ala Ala Ala Gly Ala Ala Ala
180 185 190
<210> 3
<211> 191
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> CONFLICT
<222> (1)..(191)
<223> NB41-ABD-C氨基酸序列
<400> 3
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Leu Phe Arg Ile Asn
20 25 30
Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Ser Ala Gly Ser Thr Asn Tyr Ala Asp Phe Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Thr Pro Trp Pro Val Gly Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala
115 120 125
Gln His Asp Glu Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys Val
130 135 140
Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys
145 150 155 160
Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile
165 170 175
Asp Glu Ile Leu Ala Ala Leu Pro Ala Ala Ala Cys Ala Ala Ala
180 185 190
<210> 4
<211> 573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> allele
<222> (1)..(573)
<223> NB41-ABD核苷酸序列
<400> 4
cagttgcagc tcgtggagtc tggtggaggc ttggtgcagg ctggggggtc cctgagactc 60
tcctgtgcag cctctggaag cctcttcagg atcaatgcca tggcctggtt ccgccaggct 120
ccagggaagc agcgcgagtt ggtcgcagct attactagtg ctggtagtac aaactatgca 180
gatttcgtga agggccgatt caccatctcc gcagacaacg ccaagaacac gctgtatctg 240
caaatgaaca gcctgaaacc tgaggacaca gccgtctatt actgtaatac accctggccc 300
gtagggaggg actactgggg ccaggggacc caggtcaccg tctcctcaga acccaagaca 360
ccaaaaccac aaccagcagc agctcaacat gatgaagctg ttgatgcgaa ctccctggca 420
gaagcgaaag ttctggccaa ccgtgaactg gataaatacg gcgtgtccga ctactacaaa 480
aacctgatca acaacgcaaa aactgttgaa ggcgttaagg cactgatcga tgaaattctg 540
gcagcgctgc cagcagctgc tggcgctgct gct 573
<210> 5
<211> 573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> allele
<222> (1)..(573)
<223> NB41-ABD-C核苷酸序列
<400> 5
cagttgcagc tcgtggagtc tggtggaggc ttggtgcagg ctggggggtc cctgagactc 60
tcctgtgcag cctctggaag cctcttcagg atcaatgcca tggcctggtt ccgccaggct 120
ccagggaagc agcgcgagtt ggtcgcagct attactagtg ctggtagtac aaactatgca 180
gatttcgtga agggccgatt caccatctcc gcagacaacg ccaagaacac gctgtatctg 240
caaatgaaca gcctgaaacc tgaggacaca gccgtctatt actgtaatac accctggccc 300
gtagggaggg actactgggg ccaggggacc caggtcaccg tctcctcaga acccaagaca 360
ccaaaaccac aaccagcagc agctcaacat gatgaagctg ttgatgcgaa ctccctggca 420
gaagcgaaag ttctggccaa ccgtgaactg gataaatacg gcgtgtccga ctactacaaa 480
aacctgatca acaacgcaaa aactgttgaa ggcgttaagg cactgatcga tgaaattctg 540
gcagcgctgc cagcagctgc ttgtgctgct gct 573
Claims (8)
1.一种近红外荧光纳米抗体探针，其特征在于，所述探针包括识别CEACAM5分子的纳米抗体重组蛋白和近红外荧光染料。
2.如权利要求1所述的近红外荧光纳米抗体探针，其特征在于，所述识别CEACAM5分子的纳米抗体重组蛋白选自NB41，NB41-ABD和NB41-ABD-C中的任意一个。
3.如权利要求2所述的近红外荧光纳米抗体探针，其特征在于，所述识别CEACAM5分子的纳米抗体重组蛋白NB41的氨基酸序列如SEQ ID NO:1所示。
4.如权利要求2所述的近红外荧光纳米抗体探针，其特征在于，所述识别CEACAM5分子的纳米抗体重组蛋白NB41-ABD的氨基酸序列如SEQ ID NO:2所示。
5.如权利要求2所述的近红外荧光纳米抗体探针，其特征在于，所述识别CEACAM5分子的纳米抗体重组蛋白NB41-ABD-C的氨基酸序列如SEQ ID NO:3所示。
6.如权利要求1所述的近红外荧光纳米抗体探针，其特征在于，所述近红外荧光染料选自IRDye800、IRDye680、Cy7.5、Cy5.5中的任意一个。
7.权利要求1所述的近红外荧光纳米抗体探针的制备方法，其特征在于，所述制备方法包括如下步骤：取纳米抗体重组蛋白与近红外荧光染料混合，室温避光反应2h，通过PD10层析柱进行分离,得到所述近红外荧光纳米抗体探针。
8.如权利要求1所述的近红外荧光纳米抗体探针，其特征在于，所述探针应用于检测CEACAM5分子的表达情况。
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