CN109232739A - 一种抗cd38纳米抗体、编码基因及应用 - Google Patents
一种抗cd38纳米抗体、编码基因及应用 Download PDFInfo
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- CN109232739A CN109232739A CN201710560620.4A CN201710560620A CN109232739A CN 109232739 A CN109232739 A CN 109232739A CN 201710560620 A CN201710560620 A CN 201710560620A CN 109232739 A CN109232739 A CN 109232739A
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Abstract
本发明涉及一种抗CD38纳米抗体、编码基因及应用。该抗CD38纳米抗体是如下a)或b)的蛋白：a)由序列表中序列2所示的氨基酸序列组成的蛋白质；b)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且与特异性识别CD38相关的由a)衍生的蛋白质。该抗CD38纳米抗体能够与CD38高效、特异性地结合，亲和力高达4nM，可在大肠杆菌表达系统中低成本、大规模生产，在检测和制药领域具有重要的应用前景。
Description
技术领域
本发明属于生物医学或生物制药技术领域，涉及一种抗CD38纳米抗体、编码基因及应用。
背景技术
二十世纪八十年代，CD38发现于T淋巴细胞表面，被用作细胞分化的标记物。2004年，在多发性骨髓瘤细胞中发现CD38普遍处于高表达状态，而在正常淋巴细胞、骨髓细胞以及一些非造血细胞上处于低表达状态，这使CD38成为治疗骨髓瘤的一个理想靶点。目前，有数个抗CD38的单克隆抗体正在临床使用或试验阶段，包括daratumumab、SAR650984和MOR202等。
1993年，Hamers-Casterman等人在驼科生物的血液中发现了仅由重链组成的重链抗体。而重链抗体的可变区，是能够与抗原结合的最小抗体单元，具有与重链抗体相似的抗原结合能力，称为单域抗体或纳米抗体。与传统单克隆抗体相比，单域抗体有以下优势：(1)分子量小，穿透性好；(2)有更好的溶解性和稳定性；(3)易大量生产，可以在细菌、酵母或植物中表达，所以生产成本很低；(4)不易聚集；(5)免疫原性低，更容易改造或人源化。
相对于单域抗体而言，单克隆抗体的生产成本非常高，因为它一般用哺乳细胞进行生产；另外，获得具有细胞毒活性的高亲和力抗体相对困难；且其功效的发挥还与机体的免疫状态相关，往往还因为活性不够需要与其他药物联用等缺点。而相对于单链抗体scFv而言，纳米抗体在亲和力方面与其对应的scFv相当，但在可溶性、稳定性、对聚集的抗性、可重复折叠性、表达产率以及DNA操作、文库构建和结构测定的容易性方面都优于scFv。
发明内容
本发明为了解决上述问题，提供的技术方案之一为：提供一种抗CD38纳米抗体，是如下a)或b)的蛋白：
a)由序列表中序列2所示的氨基酸序列组成的蛋白质；
b)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且与特异性识别CD38相关的由a)衍生的蛋白质。
本发明为了解决上述问题，提供的技术方案之二为：提供该抗CD38纳米抗体的编码基因。
本发明提供的所述编码基因是如下1)或2)或3)的基因：
1)其核苷酸序列是序列表中序列1；
2)在严格条件下与序列1限定的DNA片段杂交且编码与特异性识别CD38相关蛋白的DNA分子；
3)与1)或2)的基因具有90％以上的同源性，且编码与特异性识别CD38相关蛋白的DNA分子。
本发明采用PCR扩增的方法，获得了抗CD38纳米抗体的编码基因的CDS序列。其cDNA序列全长为375bp(序列1)，具体序列如下：
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACACCGATAGTGATTACATCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGTGGTCGCAACTATTTATATTGGTGGTACGTACATCCACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAATGCCGAGAACACGGTGTATCTGCAAATGAACAACCTGAAACCTGAAGACACTGCCATGTACTACTGTGCGGCCACGAAATGGCGCCCCTTTATATCGACTCGGGCAGCTGAGTATAACTACTGGGGTCAGGGGACCCTGGTCACCGTCTCCTCA
编码产生长度为125个氨基酸(末端终止子未计入，即序列中***号未计入)的蛋白质序列(序列2)，具体序列如下：
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asp Ser Asp Tyr Ile Met Ala TrpPhe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val Ala Thr Ile Tyr Ile Gly GlyThr Tyr Ile His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp AsnAla Glu Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala MetTyr Tyr Cys Ala Ala Thr Lys Trp Arg Pro Phe Ile Ser Thr Arg Ala Ala Glu TyrAsn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser***
将此抗CD38纳米抗体进行一些修饰和改造，例如进行人源化、PEG化或其他改造以提高其活性。
对此纳米抗体的CDR区域进行突变修饰，例如取代和/或缺失和/或添加一个或几个氨基酸，以提高其亲和力。
本发明为了解决上述问题，提供的技术方案之三为：含有所述编码基因的重组表达载体。
本发明为了解决上述问题，提供的技术方案之四为：包有所述编码基因的重组菌株。
本发明为了解决上述问题，提供的技术方案之五为：所述抗CD38纳米抗体、所述编码基因、所述重组表达载体、所述重组菌株在检测和制药领域中的应用。
本发明的上述应用中，包括如下中的至少一种：
1)直接应用于与病变细胞特异性地结合，例如多发性骨髓瘤和慢性淋巴白血病的病变细胞；
2)在所述抗CD38纳米抗体上连接效应分子，例如人抗体的Fc结构域、细胞因子、毒性蛋白、药物分子、放射性同位素或者载药纳米颗粒等，用于制药领域；
3)在所述抗CD38纳米抗体上添加荧光分子或化学发光基团，用于检测和追踪；
4)将所述抗CD38纳米抗体用于制作胶体金试剂盒或ELISA试剂盒，用于定量检测；
5)将所述抗CD38纳米抗体用于他免疫治疗细胞的制备，例如CAR-T细胞、CAR-DC等。
实施本发明，具有以下有益效果：我们所筛选的CD38纳米抗体具有与其他抗体不同的序列，能够与CD38高效、特异性地结合，亲和力高达4nM，而且可在大肠杆菌表达系统中低成本、大规模生产。
附图说明
为了更清楚地说明本发明实施例的技术方案，对实施例描述中所使用的附图作简单地介绍。附图中：
图1第一轮PCR产物鉴定结果；
图2第二轮PCR产物鉴定结果；
图3双酶切反应产物电泳鉴定图；
图4亲和层析电泳鉴定图；
图5离子交换层析电泳鉴定图；
图6生物膜干涉技术测量纳米抗体与CD38的亲和力。
具体实施方式
下面将结合附图对本发明的实施例进行具体描述。
由于单克隆抗体的筛选和获得难度大，而且单克隆抗体需用哺乳动物细胞进行生产导致后期成本过高。我们将CD38免疫骆驼并获得其纳米抗体的文库，通过噬菌体展示库技术，筛选获得了此nM级别亲和力的纳米抗体。此抗体可应用于多发性骨髓瘤等相关疾病的诊断及治疗，并可通过酶联吸附反应等来定性或者定量游离CD38蛋白。
实施例1 抗CD38纳米抗体噬菌体展示文库的构建及筛选
1.1 单峰驼的免疫：选取健康成年单峰驼一只，将通过酵母表达的CD38胞外域按10μg/Kg与弗氏佐剂按1:1的比例混匀，按采用背部皮下多点注射的方式免疫，共免疫六次，免疫间隔为4周。除第一次免疫使用弗氏完全佐剂，其余几次免疫均采用弗氏不完全佐剂。免疫结束后采集单峰驼颈部外周血100mL，用于构建噬菌体展示文库。
1.2 驼源淋巴细胞的分离：按照本技术领域常规程序从采集的驼源抗凝全血中分析淋巴细胞，每3×107个细胞加入1mL Trizol RNA分离液，取2mL进行RNA提取，其余-80℃保存。
1.3 总RNA提取：按eZNA Total RNA Kit(OMEGA)试剂盒说明书进行总RNA的提取。
1.4 反转录合成cDNA：
根据逆转录试剂盒TransScript Two-Step RT-PCR SuperMix(Transgene)说明书以1.3步骤获得的RNA为模板进行逆转录cDNA。
1.5 抗体可变区基因扩增：将反转录得到的cDNA作为模版进行PCR反应。扩增共进行两轮，第二轮PCR使用第二轮PCR的产物作为模版。
第一轮PCR的引物序列如下：
Primer1：5’-GTCCTGGCTGCTCTTCTACAAGG-3’
Primer2：5’-GGTACGTGCTGTTGAACTGTTCC-3’
第一轮反应条件和程序为94℃5min；94℃30s，55℃30s，72℃45s，30cycle；72℃10min
第一轮PCR产物鉴定见图1，其中，1-4：第一轮PCR产物；M：分子量标记。
第二轮PCR的引物序列如下：
Primer1：5’-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3’
Primer2：5’-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3’
第二轮反应条件和程序为94℃5min；94℃30s，60℃30s，72℃45s，30cycle；72℃10min
第二轮PCR产物鉴定见图2，其中，1-2：第二轮PCR产物；M1-2：分子量标记。
1.6 载体构建
将噬菌体载体pHEN2与第二次PCR产物分别进行NcoI和EcoRI(Thermo Fisher)双酶切，取酶切后载体5μg和酶切后的第二次PCR产物2.5μg，加T4 DNA连接酶(ThermoFisher)，16℃过夜连接并回收连接产物。图3为噬菌体载体双酶切反应产物电泳鉴定图：1-2为载体双酶切产物；M为分子量标记。
1.7 电转化及库容测定
取纯化后的连接产物，加入到含有大肠杆菌TG1电转化感受态细胞的预冷电转杯中置入电转仪(MicroPulser Electroporator，Bio-Rad)进行电转化，取出电转杯，复苏并培养转化子。随机挑选24个克隆，进行测序鉴定，并根据阳性率推算库容为2×108(库容量＝克隆数×稀释倍数×[阳性率]测序结果×10)。
引物序列如下：
MP57：5’-TTATGCTTCCGGCTCGTATG-3’
GIII：5’-CCACAGACAGCCCTCATAG-3’
1.8 噬菌体扩增
取复苏的菌液接种至2×YT-AG培养基中，37℃220rpm培养到培养物OD600＝0.5。取出菌液加入5×1010M13KO7辅助噬菌体，37℃静止感染30min。4000rpm，常温离心10min，去净上清。用2×YT-AK(含氨苄青霉素和卡那霉素)培养基重悬菌体，30℃250rpm培养过夜。离心取上清40ml管中，加入PEG8000/NaCl(20％/2.5M)溶液充分混合，离心弃上清，沉淀用1mL冰PBS洗涤离心，取上清。
测定噬菌体滴度：将TG1培养至OD600＝0.4，用LB培养基梯度稀释噬菌体，取倍比稀释的噬菌体TG1培养物混合培养，次日观察培养板中噬菌斑形成情况，对噬菌斑数在20-200的稀释梯度平板进行计数并按照下列公式计算噬菌体滴度(pfu)。
噬菌体滴度(pfu/ml)＝稀释倍数×噬菌斑数目×100
1.9 纳米抗体筛选
通过酶联免疫反应ELISA方法，以可溶解的CD38为抗原，筛选阳性克隆。将溶解在100mM NaHCO3，pH8.2的CD38抗原5μg包被ELISA板(NUNC)，5％BSA封闭，PBST(0.1％Tween-20)洗涤。每孔加入100μl噬菌体上清液，37℃放置1h。弃上清，加入150mM Glycine缓冲液pH1.5将CD38特异抗体解离出来，或者加入HRP标记的小鼠抗M13的抗体(GE Healthcare)，37℃放置1h。弃上清，加入TMB溶液，室温孵育5min，每孔加入2M硫酸终止液，用酶标仪450nm读数。
实施例2 纳米抗体在大肠杆菌中的表达和纯化
挑选噬菌体ELSIA结果阳性的克隆，提取质粒并转化至菌株BL21 DE3感受态细胞，以100mM IPTG诱导纳米抗体蛋白表达，收集菌体并破碎，使用Ni-NTA(GE Healthcare)树脂进行纯化，使用不同浓度咪唑进行洗脱和收集，将收集的样品进行还原型蛋白电泳分析，结果参见图4，M为分子量标记；1-5分别为纳米抗体菌体破碎液，穿透液、低浓度咪唑洗脱杂蛋白及洗脱目的蛋白。
并回收目的蛋白透析至低盐溶液中，进行离子交换层析，结果参见图5，M为分子量标记；1-9分别为纳米抗体上样溶液，穿透液、不同浓度的盐离子洗脱蛋白。
另外，直接将抗CD38纳米抗体的编码基因连接表达载体，转化至表达菌株，也可以实现纳米抗体的表达。
实施例3 纳米抗体与CD38亲和力测定
通过生物膜干涉技术(Octet RED96，PALL Fortebio)，我们将纳米抗体包被于生物传感器上，并测定其与目的抗原CD38的亲和力，图6和表1为测定结果。
本发明中，通过骆驼免疫、细胞分离、噬菌体文库的构建、纳米抗体的筛选，共筛选出1株抗CD38的纳米抗体。并对抗体轻链和重链基因进行分析，以确定可变区的框架区(framework regions，FR)和互补决定区(complementarity determining regions，CDR)，具体如下：
FR1:Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly
FR2：Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val Ala
FR3：Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn AlaGlu Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met TyrTyr Cys
FR4：Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
CDR1:Tyr Thr Asp Ser Asp Tyr Ile
CDR2：Thr Ile Tyr Ile Gly Gly Thr Tyr Ile His
CDR3：Ala Ala Thr Lys Trp Arg Pro Phe Ile Ser Thr Arg Ala Ala Glu TyrAsn Tyr
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。
序列表
<110> 北京大学深圳研究生院
<120> 一种抗CD38纳米抗体、编码基因及应用
<130>
<160> 2
<210> 1
<211> 125
<212> PRT
<213> 人工序列
<400> 1
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asp Ser Asp Tyr
20 25 30
Ile Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val
35 40 45
Ala Thr Ile Tyr Ile Gly Gly Thr Tyr Ile His Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Thr Lys Trp Arg Pro Phe Ile Ser Thr Arg Ala Ala Glu Tyr
100 105 110
Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 2
<211> 375
<212> DNA
<213> 人工序列
<400> 2
CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC TCGGTGCAGG CTGGAGGGTC TCTGAGACTC 60
TCCTGTGCAG CCTCTGGATA CACCGATAGT GATTACATCA TGGCCTGGTT CCGCCAGGCT 120
CCAGGGAAGG AGCGCGAGGT GGTCGCAACT ATTTATATTG GTGGTACGTA CATCCACTAT 180
GCCGACTCCG TGAAGGGCCG ATTCACCATC TCCCGAGACA ATGCCGAGAA CACGGTGTAT 240
CTGCAAATGA ACAACCTGAA ACCTGAAGAC ACTGCCATGT ACTACTGTGC GGCCACGAAA 300
TGGCGCCCCT TTATATCGAC TCGGGCAGCT GAGTATAACT ACTGGGGTCA GGGGACCCTG 360
GTCACCGTCT CCTCA 375
Claims (7)
1.一种抗CD38纳米抗体，是如下a)或b)的蛋白：
a)由序列表中序列2所示的氨基酸序列组成的蛋白质；
b)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且与特异性识别CD38相关的由a)衍生的蛋白质。
2.权利要求1所述抗CD38纳米抗体的编码基因。
3.根据权利要求2所述的编码基因，其特征在于，所述编码基因是如下1)或2)或3)的基因：
1)其核苷酸序列是序列表中序列1；
2)在严格条件下与序列1限定的DNA片段杂交且编码与特异性识别CD38相关蛋白的DNA分子；
3)与1)或2)的基因具有90％以上的同源性，且编码与特异性识别CD38相关蛋白的DNA分子。
4.含有权利要求2或3所述编码基因的重组表达载体。
5.包有权利要求2或3所述编码基因的重组菌株。
6.权利要求1所述抗CD38纳米抗体、权利要求2或3所述编码基因、权利要求4所述重组表达载体、权利要求5所述重组菌株在检测和制药领域中的应用。
7.根据权利要求6所述的应用，其特征在于，包括如下中的至少一种：
1)直接应用于与病变细胞特异性地结合；
2)在所述抗CD38纳米抗体上连接效应分子，用于制药领域；
3)在所述抗CD38纳米抗体上添加荧光分子或化学发光基团，用于检测和追踪；
4)将所述抗CD38纳米抗体用于制作胶体金试剂盒或ELISA试剂盒，用于定量检测；
5)将所述抗CD38纳米抗体用于免疫治疗细胞的制备。
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