CN112592405B - 抗人bcma纳米抗体及其制备方法和应用 - Google Patents
抗人bcma纳米抗体及其制备方法和应用 Download PDFInfo
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- CN112592405B CN112592405B CN202011424934.XA CN202011424934A CN112592405B CN 112592405 B CN112592405 B CN 112592405B CN 202011424934 A CN202011424934 A CN 202011424934A CN 112592405 B CN112592405 B CN 112592405B
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Abstract
本发明属于生物技术领域，具体涉及一种由重链抗体的可变区组成的抗人BCMA纳米抗体及其制备方法和应用，同时提供了该抗体的编码核酸分子、表达载体、宿主细胞等。本发明制备获得的一种抗人BCMA纳米抗体对人BCMA抗原具有高亲和力结合，同时具有强的抗体内吞活性，序列新颖，是新的抗BCMA纳米抗体。
Description
技术领域
本发明属于生物技术领域，具体涉及一种由重链抗体的可变区组成的抗人BCMA纳米抗体及其制备方法和应用。
背景技术
B细胞成熟抗原(B cell maturation antigen，BCMA)，也称为肿瘤坏死因子受体超家族成员17(TNFRS17)，是富含半胱氨酸胞外域的跨膜蛋白，其参与B细胞成熟、生长和存活(Madry C et al."The characterization of murine BCMA gene defines it as anew member of the tumor necrosis factor receptor superfamily".IntImmunol.1998,10(11):1693–702；Laabi Y et al."The BCMA gene,preferentiallyexpressed during B lymphoid maturation,is bidirectionally transcribed".Nucleic Acids Res.1994,22(7):1147–54；Laabi Y et al."A new gene,BCM,onchromosome 16 is fused to the interleukin 2 gene by a t(4；16)(q26；p13)translocation in a malignant T cell lymphoma".EMBO J.1992,11(11):3897–904)。BCMA是TNF超家族的两个配体的受体：APRIL(增殖诱导配体，也称为TNFSF13，TALL-2和TRDL-1；BCMA的高亲和力配体)，和B细胞活化因子BAFF(也称为BLyS，TALL-1，THANK，zTNF4，TNFSF20和D8Ertd387e；BCMA的低亲和力配体)。APRIL和BAFF是结合BCMA并促进浆细胞存活的生长因子。负调节物TACI也结合BAFF和APRIL。APRIL和BAFF与BCMA和/或TACI的协同结合激活转录因子NF-κB并增加促存活(pro-survival)Bcl-2家族成员(例如Bcl-2、Bcl-xL、Bcl-w、Mcl-1、A1)的表达和促凋亡因子(例如Bid、Bad、Bik、Bim等)的下调，因此抑制程序性细胞死亡并促进存活。该组合作用促进B细胞分化、增殖、存活和抗体产生(综述Rickert RCet al."Signaling by the tumor necrosis factor receptor superfamily in B-cellbiology and disease".Immunol Rev.2011,244(1):115-133)。
1993年，一种来源于骆驼科的新型天然抗体被发现。该抗体天然缺失轻链而只由重链组成，其重链包含两个恒定区(CH2和CH3)、一个铰链区和一个重链可变区(即抗原结合位点)，该重链可变区的相对分子质量约为15kD，仅为常规抗体的1/10，且分子高度和直径均在纳米级别，是目前可获得的最小的功能性抗体片段，因此又被称为纳米单抗(Nanobody，Nb)，即重链单域抗体VHH(variable domain of heavy chain of heavy-chainantibody)。克隆其可变区而得到的只有一个重链可变区组成的单域抗体，是目前可以得到的具有完整功能的稳定的可结合抗原的最小单位。单域抗体具有稳定性高、水溶性好、人源化简单、靶向性高、穿透性强等特点，在免疫实验、诊断与治疗中发挥着超乎想象的巨大功能。单域抗体正逐渐成为新一代抗体诊断及治疗中的新兴力量。
因此，本领域需要开发一种抗BCMA纳米抗体，尤其是具备良好的BCMA抗原结合能力，同时具有强的功能活性的抗BCMA纳米抗体。
发明内容
本发明的目的在于提供一种特异性针对人BCMA的纳米抗体，同时提供纳米抗体的编码序列、制备方法和应用。同时本发明为基于BCMA靶点的研究包括抗体偶联药物及多特异性抗体药物开发提供了研发基础。
为了达到上述目的，本发明提供了以下技术方案：
一种抗人BCMA纳米抗体，所述抗人BCMA纳米抗体包括重链抗体的可变区；
所述重链抗体的可变区包括抗原决定簇互补区(Complementarity-determiningregion，CDR)；
所述抗人BCMA纳米抗体的CDR1的氨基酸序列为SEQ ID NO：1所示；
所述抗人BCMA纳米抗体的CDR2的氨基酸序列为SEQ ID NO：2所示；
所述抗人BCMA纳米抗体的CDR3的氨基酸序列为SEQ ID NO：3所示。
所述纳米抗体的重链可变区氨基酸序列为SEQ ID NO：4所示。
一种核苷酸分子，该核苷酸分子编码所述的抗人BCMA纳米抗体；
该核苷酸分子的序列选自SEQ ID NO：5；
序列SEQ ID NO：5编码所述的纳米抗体的重链可变区。
一种表达载体，该表达载体含有所述的核苷酸分子。
一种宿主细胞，该宿主细胞含有上述表达载体。
一种抗人BCMA纳米抗体的制备方法，该制备方法包含如下步骤：
步骤1：制备含有表达所述的抗人BCMA纳米抗体的核苷酸分子的表达载体；
步骤2：用步骤1的表达载体转染真核或原核宿主细胞；
步骤3：培养步骤2转染的真核或原核宿主细胞；
步骤4：分离纯化，获得所述的抗体。
进一步的，所述的表达载体含有选自SEQ ID NO：5的核苷酸序列。
本发明还涉及包含上述的抗人BCMA纳米抗体的抗体偶联药物及多特异性抗体药物或药物组合物的开发。
本发明还提供所述抗人BCMA纳米抗体在制备抗肿瘤药物、传染性疾病或其它由BCMA介导的病理病症的药物中的应用。
相对于现有技术，本发明具有以下优点：本发明能特异识别人BCMA的纳米抗体，该抗体与人BCMA结合的亲和力强于现有抗人BCMA单克隆抗体，序列新颖。
附图说明
图1为抗人BCMA纳米抗体的第一轮PCR电泳图，M为分子量标准物(条带大小依次为2000、1500、1000、750、500、250、100bp)，“1”代表第一对引物扩增的PCR产物，“2”代表第二对引物扩增的PCR产物，PCR产物条带大小约700bp；
图2为抗人BCMA纳米抗体的第二轮PCR电泳图，M为分子量标准物(条带大小同上)，“1”代表第一对引物扩增的PCR产物，“3”代表第二对引物扩增的PCR产物，“5”代表第三对引物扩增的PCR产物，“7”代表第四对引物扩增的PCR产物，“9”代表第五对引物扩增的PCR产物，“11”代表第六对引物扩增的PCR产物，编号“2”、“4”、“6”、“8”、“10”、“12”为阴性对照孔，PCR产物条带大小约400bp；
图3为菌落PCR方法鉴定VHH片段的阳性克隆效率，M为分子量标准物(条带大小同上)，随机挑取94个单克隆(编号1-94)，编号“95”为阳性对照菌株(菌液PCR条带大小约750bp)，VHH片段的阳性克隆率约93.6％；
图4为纯化的抗人BCMA纳米抗体SDS-PAGE电泳图，图中泳道M为分子量标准物(条带大小依次为250、130、100、70、55、35、25、15、10kDa)，泳道1为阳性对照Herceptin，泳道2为纯化后的抗人BCMA纳米抗体，蛋白分子量大小约为35kDa；
图5为间接ELISA测定纳米抗体对人BCMA蛋白的结合能力；
图6为抗人BCMA纳米抗体的内吞活性检测。
具体实施方式
下文将通过实施例的方式进行本发明进一步的说明，但是本发明的保护范围不限于这些实施例。
实施例1羊驼噬菌体VHH展示文库的构建
1.1羊驼免疫
取1mg人BCMA-Fc(Acro biosystems，Cat#BC7-H5254)蛋白与弗氏完全佐剂1:1等体积混匀并充分乳化，采用皮下多点注射的方式对一只健康成年羊驼进行第一次免疫；其后用0.5mg BCMA蛋白1:1等体积混合不完全弗氏佐剂进行3次加强免疫，共免疫4次，单次间隔为20天。每次免疫7天后，抽取少量羊驼外周血进行效价检测。最后一次检测血清效价后采集外周血100ml，从该血中获得外周血单个核细胞。
1.2提取羊驼外周血单个核细胞(PBMC)总RNA以及cDNA的获得
使用FastPure Cell/Tissue Total RNA Isolation Kit(Vazyme，Cat#RC101)从保存于TRIzol里的羊驼PBMC中提取总RNA。过程简述如下，以每1ml TRIzol加入200μl氯仿的比例加入氯仿，盖好EP管盖，剧烈震荡15s，4℃静置5min。于4℃，13,000g离心10min，此时样品分为三层：红色有机相，中间层和上层无色水相，RNA主要在上层水相中。在得到的水相溶液中加入1.6倍体积Buffer RL2，轻柔混匀。混合液转移至RNAPure Columns中，13,000g室温离心1min，弃废液。向RNAPure Columns中加入500μl Buffer RW1，13,000g室温离心1min，弃废液。加入700μl Buffer RW2，13,000g室温离心1min，弃废液。再加入700μlBuffer RW2，13,000g室温离心1min，弃废液。13,000g离心2min，以彻底去除RNAPureColumns中残留的Buffer RW2。将吸附柱转移至新的RNase-free Collection Tubes 1.5ml离心管中，向吸附柱中央部位悬空滴加50μl的RNase-free ddH2O。室温放置2min，13,000g室温离心1min，洗脱RNA。
用Hiscript II 1st Strand cDNA Synthesis kit(+gDNA wiper)(Vazyme，Cat#R212-01)将总RNA反转录成cDNA步骤如下：a.RNA模板变性：在RNase-free离心管中配制如下反应体系：11μl总RNA+1μl Oligo(dT)23VN(50μM)(共12μl体系)。配置好体系，瞬时离心PCR管后，样品于65℃加热5min，迅速置于冰上骤冷，静置2min；b.基因组DNA去除：在步骤a中加入4μl 4×gDNA wiper mix，混匀后42℃，2min；c.配制第一链cDNA合成反应液：在b步骤中加入2μl 10×RT Mix+2μl Hiscript II Enzyme mix(共20μl体系)，并混匀；d.按下列条件进行第一链cDNA合成反应：50℃，45min；85℃，2min。获得的cDNA利用MiniEluteReaction Cleanup Kit(50)(QIAGEN，Cat#28204)试剂盒进行纯化。
1.3以cDNA为模板，用巢式PCR扩增得到重链抗体的可变区片段
1.3.1第一轮PCR
以上述获得的cDNA为模板，利用下表1所示的引物进行第一轮PCR扩增。
表1：羊驼噬菌体VHH展示文库构建第一轮PCR引物
第一轮PCR反应体系：0.18μl cDNA+25μl 2×Taq Master Plus Mix+0.2μl上游引物+0.2μl下游引物+24.42μl ddH2O(共50μl体系)。循环扩增条件：95℃*5min；94℃*1min，62℃*1min，72℃*1min 10s，30个循环；72℃*10min。
随后将获得的PCR产物进行琼脂糖凝胶电泳，如图1，结果显示，扩增出的VHH-CH2片段大小约700bp，即纳米抗体基因电泳条带约为700bp。
1.3.2第二轮PCR
以第一轮PCR产物为模板，利用下表2所示的引物进行第二轮PCR扩增。
表2：羊驼噬菌体VHH展示文库构建第二轮PCR引物
第二轮PCR反应体系：0.71μl VHH-CH2+25μl 2×Taq Master Plus Mix+0.2μl上游引物+0.2μl下游引物+23.89μl ddH2O(共50μl体系)。循环扩增条件：95℃*5min；94℃*1min，56℃*1min，72℃*30s，30个循环；72℃*10min。
随后将获得的PCR产物进行琼脂糖凝胶电泳，结果如图2所示，扩增出的VHH片段大小约400bp，即得到羊驼外周血液中天然缺失轻链的抗体的重链可变区片段。
1.4片段与载体的酶切、连接以及电转
使用限制性内切酶Pst I和Not I(购自NEB公司)双酶切纯化后的第二轮PCR产物VHH，使用限制性内切酶Pst I，Xba I和Not I(购自NEB公司)三酶切噬菌体载体pMECS，并以摩尔比1:5将二者的酶切产物用T4 DNA连接酶(NEB，Cat#M0202L)连接起来。具体酶切体系及连接体系如下。酶切第二轮PCR产物VHH体系：3.58μl VHH(2ng)+5μl CutSmart Buffer+1μl Pst I+1μl Not I+39.42μl ddH2O(共50μl体系)；酶切噬菌体载体pMECS体系：4.7μlpMECS+5μl CutSmart Buffer+1μl Pst I+1μl Not I+1μl Xba I+37.3μl ddH2O(共50μl体系)。酶切条件：37℃，过夜酶切。酶切结束后的片段和载体分别使用MiniElute ReactionCleanup Kit(50)(QIAGEN，Cat#28204)试剂盒进行纯化，再进行连接操作。连接反应体系如下：1μl酶切的VHH+0.81μl酶切的pMECS+1μl T4 DNA Ligase+2μl T4 DNA Ligase Buffer(10×)+15.19μl ddH2O(共20μl体系)。连接条件：先16℃，过夜连接，再在65℃，作用15min。
1.5羊驼噬菌体VHH展示文库的获得
将连接产物电转化至电转感受态细胞TG1(购自Lucigen Corporation)，电转化条件：1.8KV，200MΩ，25μF。将电转化后的菌液在SOC液体培养基中于37℃摇床培养1小时，先取100μl稀释105、106、107、108倍后，再分别取100μl涂布2×YT固体培养皿表面用于库容量测定，其余菌液涂布在2×YT固体培养皿表面，于37℃培养箱中过夜培养，次日收集所有菌落为VHH抗体文库。用菌落PCR方法鉴定VHH片段的阳性克隆效率(即VHH片段插入率)，其中菌落PCR使用的引物为：MP57：TTATGCTTCCGGCTCGTATG；GIII：CCACAGACAGCCCTCATAG，图3中结果显示VHH片段的阳性克隆率在93.6％左右，证明获得VHH抗体文库，其库容量为9.0×108。
辅助噬菌体救援步骤如下：1)取73μl的文库，接种于60ml的2×YT/Amp/Glucose(简称2YTAG)培养基中，37℃，220rpm，震荡培养至对数期OD600约为0.3～0.5；2)取出10ml培养完成的培养液，加入1×1010pfu的辅助噬菌体M13KO7(NEB，Cat#N3015S)，轻轻混匀后，于37℃静置侵染30min；3)将离心管45°斜置于培养箱中，220rpm，37℃培养1.5h左右；4)将培养液在室温，2800g，离心10min，弃上清，沉淀菌体，用50ml的2×YT/Amp/Kana培养液重悬，30℃，200rpm，培养过夜；5)将培养液于4℃，8000g，离心20min，取上清，加入1/4体积的PEG6000/2.5M NaCl，冰浴中静置2h；6)于4℃，12000g，离心15min，弃上清，沉淀以6ml左右冰浴的DPBS重悬；7)再于4℃，12000g，离心5min，取上清约6ml，加入1/4体积的PEG6000/2.5M NaCl，轻轻混合均匀，冰浴中静置2h；8)于4℃，8000g，离心20min，弃去上清，沉淀以冰浴的DPBS·20％Glycerol(0.7ml)重悬。得到重组噬菌体抗体库，分装到1.5ml EP管中，4℃保存。纯化Phage最终滴度测定步骤：准备一支干净的1.5ml EP管，取20μl噬菌体溶液，加入到80μl DPBS·20％Glycerol，混合均匀，从混合溶液中再取20μl噬菌体溶液，加入到80μlDPBS·20％Glycerol，混合均匀，以此类推，共进行5个梯度稀释，稀释后测定OD260值(即RNA浓度读值)，Phage滴度计算：[Dilution×(RNA浓度读值)]×22.14×1010pfu/ml，最终获得Phage滴度为1.1×1014pfu/ml。
实施例2：表达纳米抗体BCMA-VHH的阳性克隆株的筛选
2.1亲和淘选
制备固相蛋白ELISA板：用1x carbonate/bicarbonate buffer配制人BCMA-His抗原(购自ACRO，Cat#BCA-H522y)，使其终浓度为1μg/ml，以100μl/孔加液于96孔酶标板(暂分为A、B、C三个区，每个区有4个孔)，4℃过夜。包被结束后，用含0.05％的吐温20的PBS溶液(即1xPBST)洗板1次后，加入200μl/孔的含有2％BSA的PBST溶液，置于37℃封闭2小时。Phage封闭：取10μl纯化的Phage、16μl静注用人免疫球蛋白IgG(pH4)(购自贵州泰邦)加入到374μl含有2％BSA的PBST缓冲液中，混合均匀，使用旋转混悬仪，室温下最低转速混悬1小时。Phage固相淘选：96孔酶标板封闭结束后洗板，向A区每个孔中加入100μl已封闭的Phage溶液，B、C区标记孔中各加入100μl的PBST溶液，室温静置孵育约1小时。之后弃去B区液体，转移A区各孔上清至B区对应孔(100μl/孔)，室温静置孵育约1小时。弃去C区液体，转移B区各孔上清至C区对应孔(100μl/孔)，室温静置孵育约1小时。洗涤Phage：孵育结束后，B、C区标记孔用200μl/孔PBST溶液反复清洗22次，再用200μl/孔PBS清洗3次。洗脱并中和Phage：清洗结束后，B、C区标记孔中各加入100μl 0.1M Glycine(pH＝2.2)进行洗脱，将B、C区孔中的洗脱产物分别合并到一只新的1.5ml离心管中，混合均匀，分别加入1M Tris·HCl(pH＝9.1)进行中和。将B区中和液加入到3ml处于对数生长期的TG1(OD600为0.48)中，产生并纯化噬菌体用于下一轮的筛选，经过2轮筛选，阳性克隆将不断得到富集，从而达到了利用噬菌体展示技术筛选抗体库中BCMA特异性抗体的目的。
2.2间接Phage ELISA初步筛选抗原阳性纳米抗体
2.2.1 VHH噬菌体单克隆上清的制备
首先制备VHH噬菌体单克隆上清：挑取2轮淘筛后涂布在2YTAG平板上长出的单个菌落，接种至2块96深孔板中，该板标记为Master Plate，37℃，220rpm培养至OD600达到0.5～0.6时，转接至2块新的含有2×YT·Amp的96深孔板中，标记为P1Plate，37℃，220rpm培养至OD600值0.6～0.8。加入终浓度为1mM IPTG(QIAGEN，Cat#RT108-01)，30℃，220rpm过夜诱导；培养结束后，4000rpm室温离心15min，其上清即为VHH噬菌体单克隆上清。
2.2.2间接ELISA筛选与人BCMA抗原结合的抗体
用1x carbonate/bicarbonate buffer配制人BCMA-His抗原，使其终浓度为1μg/ml，以100μl/孔加液于96孔酶标板，4℃过夜包被。包被结束后，用PBST洗板1次后，加入200μl/孔的含有5％脱脂奶粉的PBST溶液置于37℃封闭2小时。再次洗板，加入100μl/孔的VHH噬菌体单克隆上清以及生物素标记的参照抗体人BAFF-Fc(购自Acro biosystems，Cat#BAF-H4268)。37℃孵育40min，而后洗板4次。样品孔以100μl/孔加入1：2000稀释的辣根过氧化物酶标记的抗HATag(GenScript，Cat#A01296)抗体，对照孔以100μl/孔加入1：5000稀释的辣根过氧化物酶标记的链霉亲和素(Jackson Immuno Research，Cat#016-030-084)。37℃孵育40min，而后洗板4次，拍干。添加100μl/孔的ELISA显色底物TMB(InnoReagents，Cat#TMB-S-002)，室温显色，而后用1M的硫酸溶液终止显色，测定450nm处各孔的吸光值。当样品孔OD450值大于阴性对照至少10倍视为阳性克隆株，实验结果如表3。最终选择克隆BCMA-VHH-2B11进行后续实验。
表3 抗人BCMA纳米抗体上清检测ELISA数据
实施例3：用于测定抗人BCMA纳米抗体功能活性的体外分析方法
3.1间接ELISA测定抗体的结合能力
先将克隆BCMA-VHH-2B11进行质粒的抽提，再用PEI(polysciences，Cat#24765-1)转染试剂转入Expi293F细胞。细胞上清表达的抗人BCMA纳米抗体(含有Fc tag)用ProteinA or G柱纯化标准操作规程进行纯化。获得的纯化蛋白经SDS-PAGE检测，结果如图4。
用1x carbonate/bicarbonate buffer配制人BCMA-His抗原，使其终浓度为2μg/ml，以100μl/孔加液于96孔酶标板，37℃包被2小时。包被结束后，用1xPBST洗板4次后，加入200μl/孔的含有5％脱脂奶粉的PBST溶液置于37℃封闭2小时。再次洗板，加入100μl/孔的梯度稀释抗体溶液以及BCMABenchmark1(即BCMA-BM1，其序列选用Glaxo Smith Kline公司上市抗体偶联药物GSK2857916的抗体序列，抗体在博奥信内部合成表达，其重链氨基酸为QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；其轻链氨基酸为DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC)后，37℃孵育40min，而后洗板4次。以100μl/孔加入1：5000稀释的辣根过氧化物酶标记的山羊抗人Fcγ特定片段(Jackson ImmunoResearchLaboratories,Inc.,Cat#109-036-098)，37℃孵育40min，而后洗板4次，拍干。添加100μl/孔的ELISA显色底物TMB，室温显色3-10min，而后用1M的硫酸溶液终止显色，测定450纳米处各孔的吸光值。使用GraphPad Prism软件进行数据处理，结果如图5。从图中可以看出，本发明的BCMA-VHH-2B11纳米抗体能够识别人BCMA蛋白，且结合活性略优于Benchmark1。
3.2抗人BCMA纳米抗体的内吞活性检测
DT3C蛋白能自动结合到抗体的Fc区域，并进一步发生抗体介导的内吞而起到杀伤靶细胞的原理(Miki Yamaguchi,et al."Development of a sensitive screeningmethod for selecting monoclonal antibodies to be internalized by cells".Biochemical and Biophysical Research Communications.2014,454(4):600-3)，通过DT3C可定量比较抗人BCMA纳米抗体与BCMA Benchmark1的内吞活性，步骤如下，收集处于对数生长期的人多发性骨髓瘤U266细胞(购自ATCC)，离心，弃上清，用含有15％FBS的1640(即Assay medium)重悬计数后以100μl/孔加入细胞板中，使得每孔1.5×104个细胞，在5％CO2，37℃条件下静置培养24小时。次日，用1640(即Assay buffer)稀释纳米抗体及毒素(DT3C-His)，使其工作浓度均为10μg/ml。将配置好的抗体与毒素按1:1混合，室温孵育30min。将孵育结束的混合液梯度稀释后以100μl/孔加入细胞板中，在5％CO2，37℃条件下静置培养3天。培养结束后，取出细胞板，以50μl/孔加入解冻至室温的CellCounting-Lite 2.0Luminescent Cell Viability Assay(Vazyme,Cat#DD1101-02)，室温避光放置3-5min，Tecan i-control检测荧光信号值。使用GraphPad Prism软件进行数据处理，结果如图6。从图中可以看出，本发明的纳米抗体内吞效果良好，且内吞活性优于Benchmark1。
实施例4：抗人BCMA纳米抗体的可变区测序
先将表达纳米抗体的克隆扩大培养：取出BCMA-VHH-2B11，按1：100加入2×YT培养基，37℃，200rpm培养过夜。次日，取出培养完成的菌液，使用PureYieldTM PlasmidMiniprep System试剂盒(Promega，Cat#A1223)进行质粒的抽提。步骤如下：1)取600μl菌液到1.5ml离心管中；2)加入100μl细胞裂解buffer，上下颠倒6次；3)加入350μl 4℃保存的中和溶液，并混匀；4)12,000rpm，室温离心3min；5)取上清，转移至PureYieldTM Minicolumn，并放入PureYieldTM Collection Tube；6)12,000rpm，室温离心15s；7)弃去上清，加入200μl内毒素去除液，12,000rpm，室温离心15s；8)加入400μl柱清洗液，12,000rpm，室温离心30s；9)将Minicolumn转移到新的1.5ml EP管中，加入30μl Elution Buffer，室温静置1min，12,000rpm，室温离心15s，获得质粒DNA。将抽提完成的质粒送出测序以获得纳米抗体的可变区序列，如表4。
表4 抗人BCMA纳米抗体的氨基酸序列和核苷酸序列
抗人BCMA纳米抗体的核苷酸序列如下：
BCMA-VHH-2B11纳米抗体的核苷酸序列：
5’-CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACCTTCAATAATGATAACGTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGTTATTAGTTGGAGGGGTGATGGCACGGATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACTACGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTGCAACCTAAGGACACGGCCGTTTATTACTGTGCAGTAGGCGATGGTAGCAGATGGCGCGGCGCTTATGCCTACTGGGGCCAGGGGACCCGGGTCACGGTCTCCTCA-3’(SEQID No.:5)
综上所述，本发明制备获得的抗BCMA纳米抗体(BCMA-VHH-2B11纳米抗体)对人BCMA抗原具有高亲和力结合，同时具有强的抗体内吞活性，序列新颖，是新的抗人BCMA纳米抗体。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。
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<211> 363
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
caggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggagg caccttcaat aatgataacg tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcagtt attagttgga ggggtgatgg cacggattat 180
gcagactccg tgaagggccg attcaccatc tccagagact acgccaagaa gatggtgtat 240
ctgcaaatga acagcctgca acctaaggac acggccgttt attactgtgc agtaggcgat 300
ggtagcagat ggcgcggcgc ttatgcctac tggggccagg ggacccgggt cacggtctcc 360
tca 363
Claims (10)
1.一种抗人BCMA纳米抗体，其特征在于，所述抗人BCMA纳米抗体包括重链可变区；
所述重链可变区包括互补决定区；
所述抗人BCMA纳米抗体的CDR1的氨基酸序列为SEQ ID NO：1所示；
所述抗人BCMA纳米抗体的CDR2的氨基酸序列为SEQ ID NO：2所示；
所述抗人BCMA纳米抗体的CDR3的氨基酸序列为SEQ ID NO：3所示。
2.根据权利要求1所述的抗人BCMA纳米抗体，其特征在于，所述纳米抗体的重链可变区氨基酸序列为SEQ ID NO：4所示。
3.一种核苷酸分子，其特征在于，该核苷酸分子编码如权利要求1或2所述的抗人BCMA纳米抗体。
4.根据权利要求3所述的核苷酸分子，其特征在于，所述核苷酸分子的序列选自SEQ IDNO：5；
序列SEQ ID NO：5编码所述的纳米抗体的重链可变区。
5.一种表达载体，其特征在于，该表达载体含有如权利要求3所述的核苷酸分子。
6.一种宿主细胞，其特征在于，该宿主细胞含有如权利要求5所述的表达载体。
7.一种抗人BCMA纳米抗体的制备方法，其特征在于，该制备方法包含如下步骤：
步骤1：制备含有表达如权利要求1或2所述的抗人BCMA纳米抗体的核苷酸分子的表达载体；
步骤2：用步骤1的表达载体转染真核或原核宿主细胞；
步骤3：培养步骤2转染的真核或原核宿主细胞；
步骤4：分离纯化，获得所述的抗体。
8.根据权利要求7所述的抗人BCMA纳米抗体的制备方法，其特征在于，所述的表达载体含有选自SEQ ID NO：5的核苷酸序列。
9.包括权利要求1或2所述的抗人BCMA纳米抗体的抗体偶联药物、多特异性抗体药物、嵌和抗原受体或药物组合物。
10.权利要求1或2所述的抗人BCMA纳米抗体在制备多发性骨髓瘤药物中的应用。
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