CN109970855A - Il23单域抗体的vhh链、il23单域抗体、核苷酸序列及试剂盒 - Google Patents
Il23单域抗体的vhh链、il23单域抗体、核苷酸序列及试剂盒 Download PDFInfo
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- CN109970855A CN109970855A CN201910297965.4A CN201910297965A CN109970855A CN 109970855 A CN109970855 A CN 109970855A CN 201910297965 A CN201910297965 A CN 201910297965A CN 109970855 A CN109970855 A CN 109970855A
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- domain antibody
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Abstract
本发明公开IL23单域抗体的VHH链、IL23单域抗体、核苷酸序列及试剂盒,其中,IL23单域抗体的VHH链的氨基酸序列为SEQ ID NO:1所述的氨基酸序列。本发明技术方案得到一种特异性结合IL23蛋白的单域抗体,该抗体特异性结合IL23蛋白的活性高,表达量高,成本低,易改造。
Description
技术领域
本发明涉及基因工程技术领域,特别涉及一种IL23单域抗体的VHH链、IL23单域抗体、核苷酸序列及试剂盒。
背景技术
IL-23是一种异二聚体细胞因子,是IL-12家族成员。IL-23主要由活化的树突细胞和巨噬细胞分泌,能促进T细胞的增殖及IFN-γ的产生,具有较强的抗肿瘤活性和抗感染免疫保护功能。研究表明IL-23高表达可引起多种自身免疫性疾病,如银屑病、类风湿关节炎、系统性红斑狼疮、炎症性肠病、多发性硬化、中枢神经系统性自身免疫性炎症等。
IL23拮抗剂(包括例如靶向例如IL23的p19亚基的抗IL23抗体或其抗原结合片段)可以有效降低IL-23水平,缓解因IL23过高而导致的免疫紊乱症状。例如延迟或预防IL23介导的疾病或病症的发作,减少IL23介导的疾病或病症的发生。
然而,现有技术中的IL-23抗体活性较低,表达量也很低。
发明内容
本发明的主要目的是提供一种多肽序列,旨在解决提高IL23单域抗体的活性。
为实现上述目的,本发明提供一种IL23单域抗体的VHH链,所述VHH链的氨基酸序列为SEQ ID NO:1所述的氨基酸序列。
本发明还提供一种IL23单域抗体,所述IL23单域抗体包含SEQ ID NO:1所述氨基酸序列。
本发明还提供一种核苷酸序列,其特征在于,所述核苷酸序列编码SEQ ID NO:1所述的氨基酸序列。
在一实施例中,所述核苷酸序列如下:
CAGGTAAAGCTGGAGGAGTCTGGGGGAGGATTGGTACAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATGGAACCTTGGTAATTATGCCTTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATCGACTGGCGTCATAGTTCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACATGGTGTATCTGCAAATGAGCAGCCTGAAACTTGAGGACACGCGCCTTTATTACTGTGCAGCATCAAGCCTATTCCCTAGTAGTGCTCCCCGTCAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
本发明还提供一种试剂盒,所述试剂盒包括IL23单域抗体的VHH链,或者包含核苷酸序列;所述VHH链的氨基酸序列为SEQ ID NO:1所述的氨基酸序列;所述核苷酸序列为:CAGGTAAAGCTGGAGGAGTCTGGGGGAGGATTGGTACAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATGGAACCTTGGTAATTATGCCTTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATCGACTGGCGTCATAGTTCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACATGGTGTATCTGCAAATGAGCAGCCTGAAACTTGAGGACACGCGCCTTTATTACTGTGCAGCATCAAGCCTATTCCCTAGTAGTGCTCCCCGTCAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
本发明技术方案通过结合基因工程的方法得到一种特异性结合IL23蛋白的单域抗体,该抗体特异性结合IL23蛋白的活性高,表达量高,成本低,易改造。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1为本发明第一轮PCR单域抗体基因电泳图;
图2为图1中1道中750bp-500bp,2道和3道DNA回收后进行第二轮PCR单域抗体基因电泳图;
图3为蛋白表达纯化图;
图4为本发明IL23单域抗体与IL23抗原结合活性图。
图5为本发明IL23单域抗体亲和力检测过程图;
图6为本发明IL23单域抗体亲和力检测过程与线性拟合对比图。
本发明目的的实现、功能特点及优点将结合实施例,参照附图做进一步说明。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实提出了一种IL23单域抗体、核苷酸序列及试剂盒。下述内容将针对所述多肽及其筛选过程作详细介绍。
首先,本发明的多肽具有三个框架区和两个可变区:
FR1:Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Trp Asn Leu Gly;
CDR1:Asn Tyr Ala Leu Gly;
FR2:Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala;
CDR2:Ala Ile Asp Trp Arg His Ser Ser Tyr Tyr Ala Asp Ser Val Lys;
FR3:Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Met Val Tyr LeuGln Met Ser Ser Leu Lys Leu Glu Asp Thr Arg Leu Tyr Tyr Cys Ala;
CDR3:Ala Ser Ser Leu Phe Pro Ser Ser Ala Pro Arg Gln Tyr Asp;
FR4:Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser。
针对该IL23单域抗体,本发明构建方式分为抗体库的构建、特异性噬菌体的筛选、特异性阳性单克隆的筛选、IL23单域抗体在宿主大肠杆菌中表达、纯化。下述内容将针对每一步进行详细阐述。
一、抗体库的构建
IL23抗原:厂家北京义翘神州,货号CT048-H08H。
使用2mg上述抗原与等体积福氏佐剂混合。选择成年健康的羊驼,注射该抗原,分9次免疫,第4次免疫后,采取羊驼血清,通过酶联免疫的方法,测定抗原免疫效价。
a0:分离淋巴细胞。当免疫效价达到1万倍以上时,采全血150ml,使用QIAGEN试剂盒(QIAamp RNA Blood Mini Kit(50),货号,52304)将淋巴细胞分离。
b0:裂解。将分离后的淋巴细胞裂解,得到CDNA,使用QIAGEN试剂盒(QIAamp RNABlood Mini Kit(50),货号,52304),测定得到CDNA浓度。
c0:巢式PCR扩增。用cDNA合成试剂盒(MiniBESTAgarose Gel DNAExtraction KitVer.4.0,TAKARA公司),采用巢式PCR方法,进行两轮PCR扩增抗体重链可变区VHH基因片段;
第一轮PCR扩增,可得到大于800bp的普通抗体基因片段,800bp~500bp之间的为缺失轻链的重链抗体基因片段,以及500bp的重链抗体可变区片段VHH.通过电泳,筛选出800bp~500bp的基因片段和500bp的基因片段。
d0:切胶回收,将步骤c0中800bp~500bp的基因片段切胶回收。具体请参阅图1,1号带为普通抗体DNA和重链抗体DNA,能够看到其中的两条亮带,有大于800bp的(普通抗体DNA),也有在500bp~750bp之间的(重链抗体DNA),将该图中位于750bp~500bp的条带切胶回收;2号带为重链抗体可变区片段VHH,大小在500bp;将2号带目的条带也进行回收。
e0:VHH目的基因扩增。以回收的完整重链抗体及其重链可变区的基因片段为模板,用VHH特异性引物经第二轮PCR扩增,得到VHH目的基因(500bp)。请参阅图2,可以看到一条亮带,VHH目的基因大约为500bp,也就是该亮带中混杂有多种500bp左右的VHH目的基因。
上述第一轮PCR引物包括SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3核苷酸序列。
其中SEQ ID NO:1和SEQ ID NO:2配对使用,扩增得到图1中1道所示的两个条带;SEQ ID NO:2和SEQ ID NO:3配对使用,扩增得到图1中2道所示的一条条带。
第二轮PCR引物包括SEQ ID NO:4和SEQ ID NO:5核苷酸序列。SEQ ID NO:4和SEQID NO:5配对使用,得到图2所示的500bp目的基因。
基因序列名称 | 引物序列 |
SEQ ID NO:1 | CGCCATCAAGGTACCAGTTGA |
SEQ ID NO:2 | CGGGATCCCAGGTACAGCTGGTGGAGTCTGGGGGAG |
SEQ ID NO:3 | CCGCTCGAGTACTTCATTCGTTCCTGAGGAGACGGT |
SEQ ID NO:4 | CCGCTCGAGTGAGGAGACGGTGACCT GG |
SEQ ID NO:5 | CGGGATCCGAGGTACAGCTGGTGGAGTCTGGGGGAG |
f0:VHH片段转入TG1感受态细胞。将上述得到的VHH片段连接到pHEN6噬菌体展示载体质粒(通过BamHI,XhoI双酶切),之后将VHH片段及pHEN6载体(ZL20111028003.1)经连接酶连接,电转化至TG1感受态细胞中,然后将感受态细胞涂布平板,经菌落PCR验证VHH基因插入率。
当验证VHH基因插入成功后,对重组基因进行克隆效率检测:取电转化菌液涂布LB/Amp平板上,32℃,过夜培养,次日用菌落PCR的方法验证抗体的连接效率。
其中,菌落PCR的方法如下:1、用高压灭菌的牙签或枪头挑取单个菌落,先在抗性平板上点单克隆保存(标记),然后置于20ul Triton-x100(或去离子水)中搅和。2、将装有20ul Tritonx-100的EP管在100℃下煮2分钟。3、取1ul上清为模板,加入PCR体系进行PCR反应,PCR体系可以为20ul。4、琼脂糖凝胶电泳观察结果。
当噬菌体抗体库的连接效率低于90%时,说明在操作失误,需要重复上述实验过程;当噬菌体抗体库的效率达到90%时,进行下一步操作。
g0:扩大培养,保藏。将电转化菌液涂布LB/Amp平板上,32℃,过夜培养物用2YT培养基洗下,以1:1000比例在2YT培养基下进行扩大培养,加入辅助噬菌体M13K07(Invitrogen)进行感染,过夜培养,离心,收集上清后加入20%PEG-2.5M NaCl混匀(噬菌体在上清中),离心收集沉淀,加入PBS和甘油进行重悬,并保藏于-80℃备用。
二、特异性噬菌体的筛选
由于经过巢式PCR扩增出来的VHH片段有多种,而这些片段中,并非所有这些基因片段都是目标片段,将这些片段VHH片段转入噬菌体后,需要对目标噬菌体进行纯化,下述内容为纯化目标噬菌体的步骤:
a1:CPBS溶液的配备。将少量无脂牛奶加入到PBS溶液中,其中,无脂牛奶的占比在1%-5%(封闭作用);将溶解在CPBS溶液中的IL23蛋白稀释至150μg/ml;
b1:将IL23蛋白稀释液后,150μl/孔进行包被;
c1:静置,并弃包被液,加入封闭液(1%CPBS)300μl/孔,37℃封闭2h;
d1:将筛选出来的噬菌体加入到微孔中,并加入封闭液混匀至每孔体积150μl;
e1:室温孵育2h(噬菌体的外壳上分泌抗体,抗体与IL23蛋白结合);
f1:筛孔分别用PBST(含0.05%Tween20)、PBS各洗涤10次,每次2min,将没有结合的噬菌体洗掉;
g1:加入TEA到筛孔中洗脱噬菌体,吹吸混悬均匀,室温静置10min;
h1:再次吹吸混悬均匀后加入到预冷的1M Tris-HCl中混匀,进行滴度的测定;
i1:扩增并纯化扩增后的噬菌体。
上述步骤a1至步骤i1,重复三轮,并以步骤i1的噬菌体作为下一轮步骤d1中的加入微孔的噬菌体(第一轮的噬菌体来源于上述-80℃保藏备用的),(第二轮筛选包被浓度为10μg/ml,第三轮筛选包被浓度为1μg/ml。二、三轮各150μl/孔进行包被)。
筛选结果详见下表
筛选次数 | 加入噬菌体抗体库总量 | 洗脱液+Tris-HCl | 洗脱滴度 |
第一轮 | 5.60E+11 | 300ulTEA+200ulTris | 28/ul |
第二轮 | 5.20E+11 | 150ul*5TEA+350ulTris | 6.76E+3/ul |
第三轮 | 5.03E+11 | 150ul*5TEA+350ulTris | >1E+4/ul |
上述步骤a1至步骤i1以包被IL23抗原作为靶标,采用固相筛选法从总噬菌体抗体库进行3轮筛选,检测每轮洗脱下来的噬菌体滴度,由上表可以看出,随着筛选轮数的增加,包被浓度逐级减少,但洗脱下来的噬菌体滴度增加,也就是说IL23特异性噬菌体得到了高效富集。
三、特异性阳性单克隆的筛选
虽然上述噬菌体已经得到了高效富集,但是任然残留有少量非特异性的噬菌体,在在下述内容中,将进一步纯化特异性IL23单域抗体基因。具体步骤如下:
a2:通过SEQ ID NO:4和SEQ ID NO:5核苷酸序列,对上述已经富集的IL23特异性噬菌体进行PCR扩增,获得的特异性IL23单域抗体基因(带有限制性内切酶BbsI和BamHI位点的PCR产物);
b2:用限制性内切酶BbsI和BamHI分别处理PCR产物和pSJF2载体(ZL201110280031),经T4连接酶连接重组,而获得能在大肠杆菌中高效表达的质粒sdAb-pSJF2;
c2:从生长菌落的琼脂平板上随机挑取多个单菌落,然后接种在含有Amp的2YT液体培养基的96孔深孔培养板中;
d2:培养4小时后,将单克隆一一对应接种在带编号的以小格分隔的含Amp的LB固体平板上;
e2:向深孔培养板加入IPTG至终浓度0.5mM诱导;
f2:培养过夜后,收获表达蛋白的菌上清液;
g2:以IL23抗原进行ELISA测定,选出Anti-IL23阳性克隆ELISA测定结果;
h2:挑选出的IL23阳性克隆,经DNA测序以鉴定抗IL23单域抗体克隆的基因序列SEQ ID NO:6(或者与SEQ ID NO:6互补的SEQ ID NO:7)。
SEQ ID NO:6序列如下:
CAGGTAAAGCTGGAGGAGTCTGGGGGAGGATTGGTACAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATGGAACCTTGGTAATTATGCCTTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATCGACTGGCGTCATAGTTCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACATGGTGTATCTGCAAATGAGCAGCCTGAAACTTGAGGACACGCGCCTTTATTACTGTGCAGCATCAAGCCTATTCCCTAGTAGTGCTCCCCGTCAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
四、IL23单域抗体在宿主大肠杆菌中表达、纯化
得到上述阳性单克隆后,需要将其表达方可得到IL23抗体,后续主要是通过大肠杆菌表达,然后纯化,即可得到所需的IL23单域抗体。具体操作过程如下:
a3:将上述含有质粒IL23的菌种接种在含氨基苄青霉素的LB培养板上,37℃过夜。在此,由于pSJF2载体自身对氨基苄青霉素具备抗性,从而,在含氨基苄青霉素的LB培养板上,只有含有pSJF2载体的大肠杆菌能生长,避免了其它杂菌的干扰;
b3:挑选单个菌落接种于5ml的含氨基苄青霉素的LB培养液中,37℃,摇床培养过夜;
c3:转种2ml过夜培养物于200mL含氨基苄青霉素的LB培养液中;
d3:37℃摇床培养,240转/分,培养到OD值达0.4~0.6时,加入0.5~1.0mM IPTG,继续培养过夜,然后离心,收菌。
e3:高渗法裂解细菌,离心,收上清中可溶性单域抗体蛋白;
f3:经Ni+离子亲和层析获得纯度达95%以上的蛋白。
(图3与上一案例不同,需要详细分析)具体请参照图3,在图3中,M为蛋白分子标准,条带1为破菌后总蛋白粗提样品。
条带2为总蛋白粗提过镍柱后的样品,说明只有少量的样本被洗脱下来,Ni+柱中还剩余有大量的样本蛋白。
条带3为用含有40毫摩咪唑的洗脱过脱镍柱后剩余的样品,说明经过进一步洗脱后,大部分蛋白都被洗脱下来。
条带4为用含有100毫摩咪唑的洗脱液过脱镍柱后剩余的样品,说明Ni+柱中,没有被洗脱下来的蛋白极少。
条带5为用含有400毫摩咪唑的洗脱液过镍柱后剩余的样品,通过此条带可以看出,Ni+柱的蛋白几乎全部被洗脱下来,洗脱下来的目标蛋白较纯。五、单域抗体与IL23抗原结合活性测定
上述过程已经将目标抗体筛选并纯化出来,为了验证目标抗体的活性,实验步骤如下:
a4:以0.05M Na2CO3·NaHCO3(pH9.5)稀释IL23抗原至2μg/ml,100μl/孔,抗原包被96孔板,4℃孵育过夜;
b4:用PBS洗板三次,300μl2%BSA(或1%CPBS)封闭96孔板,37℃,孵育2小时;
c4:加入不同稀释浓度的纯化的IL23单域抗体,按100μl/孔加入,37℃,孵育1小时;
d4:用0.05%PBST洗板三次;
e4:加5000倍稀释的antiMyctag antibody(HRP),按100μl/孔加入,37℃,孵育1小时;
f4:用0.05%PBST洗板三次,加TMB100μl/孔,避光室温静置10分钟。g4:加入2MH2SO4 50μl/孔终止反应;
g4:用酶标仪测定450nm波长下的样品OD值。
从图4可以看出,即便IL23单域抗体与IL23抗原结合后的浓度在8ng/ml时,依然可以检测到较高的活性。
图5为亲和力检测过程图与测算结果。其中,四条曲线分别为使用不同浓度梯度抗体测得结果(1号线对应浓度为235.3nM的抗体,2号线对应浓度为470.6nM的抗体,3号线对应浓度为941.2nM的抗体,4号线对应浓度为1882nM的抗体)。横坐标为时间轴(0-900秒),纵坐标为实验过程中机器读取的反应数值(Response)。根据实验设计,300秒前为抗原抗体结合过程,300秒-900秒为解离过程,参照图5中的分割线。根据结合过程中机读数值随时间的变化,可得结合常数Kon(1/Ms)。根据解离过程中机读数值随时间的变化,可得解离常数Kdis(1/s)。根据公式:
亲和力常数KD=解离常数Kdis/结合常数Kon,可得使用不同浓度测得的IL17RA抗体亲和力常数KD=1.30E-7M。各浓度梯度下检测结果一致,实验结果可靠(参照图6中拟合曲线及下表,R2=0.999)。
对于IL IL23抗体生产效率,本发明使用分光光度计对纯化后得到的IL17RA抗体进行浓度测定,测得经表达、提取、纯化后蛋白总量为4.80mg。因实施例中所用表达体系为200ml,因此,本实施例中构建的IL IL23单域抗体原核表达系统单位表达量为2.40mg/100ml菌液。
通过分析计算最终获得的表达量,可以证明实施例中所用的表达体系对蛋白的表达效率,即单位表达体积可获得的蛋白质量。通常情况下,表达量达到0.5mg/100ml,即认为体现出了较高的表达水平(相对于现有技术中的最高水准),本次IL23单域抗体的表达量达到了2.40mg/100ml,远高行业水准。
以上所述仅为本发明的优选实施例,并非因此限制本发明的专利范围,凡是在本发明的发明构思下,利用本发明说明书及附图内容所作的等效结构变换,或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。
SEQUENCE LISTING
<110> 深圳普瑞金生物药业有限公司
<120> IL23单域抗体的VHH链、IL23单域抗体、核苷酸序列及试剂盒
<130> 123
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 309
<212> PRT
<213> 人工序列
<400> 1
Gly Leu Asn Val Ala Leu Leu Tyr Ser Ser Glu Arg Gly Leu Tyr Gly
1 5 10 15
Leu Tyr Gly Leu Tyr Val Ala Leu Gly Leu Asn Ala Leu Ala Gly Leu
20 25 30
Tyr Gly Leu Tyr Ser Glu Arg Ala Arg Gly Ser Glu Arg Cys Tyr Ser
35 40 45
Ala Leu Ala Ala Leu Ala Ser Glu Arg Gly Leu Tyr Thr Arg Pro Ala
50 55 60
Ser Asn Gly Leu Tyr Ala Ser Asn Thr Tyr Arg Ala Leu Ala Gly Leu
65 70 75 80
Tyr Thr Arg Pro Pro His Glu Ala Arg Gly Gly Leu Asn Ala Leu Ala
85 90 95
Gly Leu Tyr Leu Tyr Ser Ala Arg Gly Pro His Glu Val Ala Leu Ala
100 105 110
Leu Ala Ala Leu Ala Ile Leu Glu Ala Ser Pro Thr Arg Pro Ala Arg
115 120 125
Gly His Ile Ser Ser Glu Arg Ser Glu Arg Thr Tyr Arg Thr Tyr Arg
130 135 140
Ala Leu Ala Ala Ser Pro Ser Glu Arg Val Ala Leu Leu Tyr Ser Gly
145 150 155 160
Leu Tyr Ala Arg Gly Pro His Glu Thr His Arg Ile Leu Glu Ser Glu
165 170 175
Arg Ala Arg Gly Ala Ser Pro Ala Ser Asn Thr His Arg Leu Tyr Ser
180 185 190
Ala Ser Asn Met Glu Thr Val Ala Leu Thr Tyr Arg Gly Leu Asn Met
195 200 205
Glu Thr Ser Glu Arg Ser Glu Arg Leu Tyr Ser Ala Ser Pro Thr His
210 215 220
Arg Ala Arg Gly Thr Tyr Arg Thr Tyr Arg Cys Tyr Ser Ala Leu Ala
225 230 235 240
Ala Leu Ala Ser Glu Arg Ser Glu Arg Pro His Glu Ser Glu Arg Ser
245 250 255
Glu Arg Ala Leu Ala Ala Arg Gly Gly Leu Asn Thr Tyr Arg Ala Ser
260 265 270
Pro Thr Tyr Arg Thr Arg Pro Gly Leu Tyr Gly Leu Asn Gly Leu Tyr
275 280 285
Thr His Arg Gly Leu Asn Val Ala Leu Thr His Arg Val Ala Leu Ser
290 295 300
Glu Arg Ser Glu Arg
305
<210> 2
<211> 21
<212> DNA
<213> 人工序列
<400> 2
cgccatcaag gtaccagttg a 21
<210> 3
<211> 29
<212> DNA
<213> 人工序列
<400> 3
cgggatccca ggtacagctg gtggagtctg ggggag 36
<210> 4
<211> 43
<212> DNA
<213> 人工序列
<400> 4
ccgctcgagt acttcattcg ttcctgagga gacggt 36
<210> 5
<211> 41
<212> DNA
<213> 人工序列
<400> 5
ccgctcgagt gaggagacgg tgacctgg 28
<210> 6
<211> 47
<212> DNA
<213> 人工序列
<400> 6
cgggatccga ggtacagctg gtggagtctg ggggag 36
<210> 7
<211> 366
<212> DNA
<213> 人工序列
<400> 7
caggtaaagc tggaggagtc tgggggagga ttggtacagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggatg gaaccttggt aattatgcct tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcagct atcgactggc gtcatagttc atactatgca 180
gactccgtga agggccgatt caccatctcc agagacaaca ccaagaacat ggtgtatctg 240
caaatgagca gcctgaaact tgaggacacg cgcctttatt actgtgcagc atcaagccta 300
ttccctagta gtgctccccg tcagtatgac tactggggcc aggggaccca ggtcaccgtc 360
tcctca 366
Claims (5)
1.一种IL23单域抗体的VHH链,其特征在于,所述VHH链的氨基酸序列为SEQ ID NO:1所述的氨基酸序列。
2.一种IL23单域抗体,其特征在于,所述IL23单域抗体包含如权利要求1所述的SEQ IDNO:1氨基酸序列。
3.一种核苷酸序列,其特征在于,所述核苷酸序列编码如权利要求1所述所述的IL23单域抗体。
4.如权利要求3所述的核苷酸序列,其特征在于,所述核苷酸序列如下:
CAGGTAAAGCTGGAGGAGTCTGGGGGAGGATTGGTACAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATGGAACCTTGGTAATTATGCCTTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATCGACTGGCGTCATAGTTCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACATGGTGTATCTGCAAATGAGCAGCCTGAAACTTGAGGACACGCGCCTTTATTACTGTGCAGCATCAAGCCTATTCCCTAGTAGTGCTCCCCGTCAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
5.一种试剂盒,其特征在于,包括如权利要求1所述的IL23单域抗体的VHH链,或者包含权利要求2所述的IL23单域抗体,或者包含如权利要求3或4所述的核苷酸序列。
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