CN112553167B - 人源化抗基孔肯雅病毒nsp1抗体及其应用 - Google Patents
人源化抗基孔肯雅病毒nsp1抗体及其应用 Download PDFInfo
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Abstract
人源化抗基孔肯雅病毒NSP1抗体及其应用，所述抗体的轻链可变区具有SEQ ID NO:1核苷酸序列和SEQ ID NO:2氨基酸序列，其中轻链超变区序列具有SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5；重链可变区具有SEQ ID NO:6核苷酸序列和SEQ ID NO:7氨基酸序列，重链超变区具有SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10。该抗体能够特异性结合基孔肯雅病毒的NSP1抗原。
Description
技术领域
本发明属于基孔肯雅病毒治疗预防领域，具体涉及人源化抗基孔肯雅病毒NSP1抗体及其应用。
背景技术
基孔肯雅病毒(Chikungunya virus,CHIKV)是一种单股正链RNA病毒，可致基孔肯雅热，虽致死率不高，但被感染人群丧失劳动力，导致严重的社会影响与经济损失。CHIKV是一种蚊媒病毒，主要由埃及伊蚊和白蚊伊蚊传播给人类，1952-1953年在坦桑尼亚首次分离，随后在非洲和亚洲地区爆发流行。2005年后CHIKV再次在全球多地数次爆发，并在法国、意大利、中国出现散在的输入性病例，特别是CHIKV东中南非型E1蛋白发生突变(E1-A226V)使其可有效通过白蚊伊蚊传播，不仅对热带和亚热带地区，还对白蚊伊蚊广泛存在的温带地区的民众构成了潜在威胁。
CHIKV感染患者染病早期主要表现为发热、头痛、肌痛、皮疹和乏力等非特异性临床症状，与登革病毒(dengue virus,DENV)、寨卡病毒(Zika virus,ZIKV)、黄热病毒(yellow fever virus,YFV)、西尼罗病毒(west nile virus,WNV)感染早期症状相似，且几种病毒均以蚊子作为传播媒介，存在混合感染。这几种病毒感染的治疗方法不同，尤其是对CHIKV有效非甾体类抗炎药被严禁用于DENV感染，在CHIKV防控中，接种疫苗是最有效的保护人类远离疾病及限制CHIKV传播的手段之一，但是目前没有获得许可的针对CHIKV的疫苗和抗病毒药物。对CHIKV感染的急性期患者而言，休息、预防脱水、止痛和消炎等对症治疗是唯一可用和推荐的治疗方法。如广谱抗病毒药物利巴韦林对CHIKV感染的治疗有效，这些抗病毒药物可以减少感染者体内的病毒含量，降低症状的严重程度和减少病毒的传播，但是还需要更多的临床治疗结果来证实。目前尽管一些候选疫苗在进行评估，但尚没有一种疫苗获得批准。因此，基于目前尚无有效疫苗和抗病毒药物的形式，我们必须通过不同的策略研究和开发具有快速、特异等优点的免疫学治疗方法。
CHIKV属于披膜病毒科中的甲型病毒属，有包膜，直径60～70nm。内含一个长约12kb的单股正链RNA，编码6个结构蛋白(C-E3-E2-6k/TF-E1)和4个非结构蛋白(NSP1、NSP2、NSP3、NSP4)。非结构蛋白主要与病毒的复制过程有关，通常CHIKV首先感染宿主皮肤的细胞，包括皮肤成纤维细胞和皮肤巨噬细胞，其中成纤维细胞时CHIKV感染的主要靶细胞，病毒进入机体后大量复制，与机体的各类细胞相互作用，诱导产生各种致病因子，参与致病过程。病毒分子致病机制研究表明，CHIKV的NSP2能够阻断Ⅰ型IFN信号，导致宿主抗病毒介质减少，这有助于病毒在机体内大量扩增，同时NSP1蛋白也有助于CHIKV的复杂，这可能是CHIKV的一种免疫逃逸机制。因此，通过抑制病毒NSP2蛋白和NSP1蛋白表达有可能控制CHIKV感染。
申请人前期以基孔肯雅病毒非结构蛋白NSP1抗原制备单克隆抗体及其人源化抗体，可作为一种方便、快速而有效的诊断与治疗手段。
发明内容
解决的技术问题：本发明提供一种人源化抗基孔肯雅病毒NSP1抗体及其应用，通过单克隆抗体技术筛选到了对NSP1具有高度亲和力的单克隆抗体，并获取了赋予所述抗体异特性的重链和轻链可变区的氨基酸序列和核苷酸序列，获得了具有特异的抗原结合性抗体，可用于基孔肯雅病毒的诊断与治疗。
技术方案：抗基孔肯雅病毒NSP1蛋白(Chikunguya virus,CHIKV)的杂交瘤细胞株CH001。
抗NSP1单克隆抗体由权利要求1所述杂交瘤细胞株CH001产生。
上述单克隆抗体的重链可变区具有SEQ ID NO:2的氨基酸序列，轻链可变区具有SEQ ID NO:4的氨基酸序列。
上述单克隆抗体的重链恒定区具有SEQ ID NO:6的氨基酸序列，轻链恒定区具有SEQ ID NO:8的氨基酸序列。
上述重链可变区具有SEQ ID NO:1的DNA序列，轻链可变区具有SEQ ID NO:3的DNA序列。
上述重链恒定区具有SEQ ID NO:5的DNA序列，轻链恒定区具有SEQ ID NO:7的DNA序列。
一种含有单克隆抗体重链或轻链的核苷酸编码的表达载体，重链可变区具有SEQID NO:1的DNA序列，轻链可变区具有SEQ ID NO:3的DNA序列，重链恒定区具有SEQ ID NO:5的DNA序列，轻链恒定区具有SEQ ID NO:7的DNA序列。
上述载体为抗体IgG1亚型分泌型真核表达载体。
一种含有所述表达载体的宿主细胞，宿主细胞为FreeStyleTM293-F cells。
上述抗NSP1单克隆抗体在制备基孔肯雅病毒诊断与治疗试剂中的应用。
有益效果：发明提供了一种具有高特异性、高亲和力抗NSP1的单克隆抗体CH001。NSP1是基孔肯雅病毒表达的非结构蛋白，因此本发明的单克隆抗体NSP1可应用于有关基孔肯雅病毒的诊断、治疗及预防研究中。本发明以NSP1为靶分子，制备出鼠源单抗CH001，免疫学检测显示，该单克隆抗体能够与基孔肯雅NSP1抗原特异性结合。
附图说明
图1为NSP1质粒构建图，M：marker；1：NSP1-pET28a表达质粒；2：EcoRI-NotI双酶切；
图2为NSP1抗原表达和纯化的SDS-PAGE检测结果，A)M，marker；1，未诱导，3，小量诱导上清；2,4，小量诱导沉淀。B)M，marker；1，穿流；2-4，不同浓度咪唑洗脱液；
图3为抗NSP1单克隆抗体的SDS-PAGE检测结果，可见NSP1抗体的纯度很高；
图4为抗NSP1单克隆抗体的酶联免疫吸附试验检测结果，可见NSP1抗体与抗原的结合能力较强；
图5为hmCH001重组表达质粒构建的核酸电泳结果，逆转录PCR扩增CH001重链和轻链可变区基因。M，DL2000；1，CH001重链可变区VH；2，CH001轻链可变区VK；
图6为hmCH001重组表达质粒构建的核酸电泳结果，M，DL10000；1，重组表达hmCH001重链质粒即pFUSE-CHIg-hG1—hmCH001；2，线性化重链模板质粒pFUSE-CHIg-hG1；3，hmCH001重链可变区VH；4，hm CH001轻链质粒即pFUSE-CLIg-hk-hm CH001K；5，线性化轻链模板质粒pFUSE-CLIg-hk；6，hm CH001轻链可变区即hm CH001VK；
图7为纯化抗体hmCH001的SDS-PAGE检测结果，M，marker；1，hmCH001纯化抗体；2，对照IgG；3，培养上清；4，流穿；5，未转染质粒293F培养上清；
图8为抗NSP1单克隆抗体hm CH001的酶联免疫吸附试验检测结果；
具体实施方式
实施例1鼠源单抗CH001的制备及筛选检测
1)应用杂交瘤技术制备抗NSP1单克隆抗体，具体步骤如下：
一、制备抗原(NSP1重组蛋白)
构建NSP1表达质粒：
PCR扩增基因序列，
(F(5’-3’)：TAGAGCTAGCGAATTCGAATTCCACCATGGAGACCGATACC；
R(5’-3’)：TTGCGGCCGCGGATCCGCGGCCGCTTATCACTTGC)，限制性内切酶EcoRI和NotI分别酶切目的片段和表达质粒pET28a，胶回收目的片段和载体片段，按比例16℃连接过夜，转化大肠杆菌DH5α感受态细胞，通过菌落PCR筛选鉴定阳性克隆，提取质粒进行双酶切鉴定和测序。
将酶切和测序正确的质粒重新转化大肠杆菌BL-21(DE3)感受态细胞，筛选阳性转化子进行原核表达，对表达产物进行SDS-PAGE检测，获得了43kDa的重组蛋白，与NSP1蛋白的分子量大小相符，用His亲和层析柱(美国Amersham公司)对目的蛋白进行纯化，获取带有His标签的NSP1重组蛋白，命名为NSP1蛋白。表达质粒的构建见图1。镍柱纯化的NSP1蛋白SDS-PAGE检测见图2。
二、制备抗NSP1的杂交瘤细胞株(所有的供杂交瘤技术用的小鼠骨髓瘤SP2/0细胞系均来源于纯系BALB/c小鼠)：
用NSP1蛋白作为免疫原在小鼠腹部皮下注射进行免疫接种，每次100μg/mL，共五次，在最后一次免疫接种前三天进行腹腔内加强免疫。融合当天取小鼠脾脏，用RPMI-1640不完全培养基(美国GIBCO公司)制备成单细胞悬液，在50％PEG(pH 8.0)存在下，将脾细胞和SP2/0小鼠骨髓瘤细胞进行融合，用HAT选择性培养基(RPMI-1640培养基98mL，HT贮存液1mL，A贮存液1mL)培养7天，换用HT培养基(RPMI-1640培养基99mL，HT 1mL)培养。
三、抗NSP1阳性单克隆抗体的筛选
根据杂交瘤细胞生长情况行常规ELISA检测筛选，取检测阳性孔的细胞再次克隆化，经过5次克隆化，待所有的孔内单克隆细胞检测都为抗NSP1阳性后，取数孔扩大培养并部分冻存。最终选取1株稳定分泌抗NSP1抗体的杂交瘤细胞，分泌的单抗命名为CH001。
四、单克隆抗体的亚型鉴定
用美国Sigma公司的单克隆抗体亚型鉴定试剂盒(ISO-2KT)进行抗体的亚型，结果显示抗体亚型为IgG1。
五、单抗的大量培养并纯化
1.用杂交瘤专用无血清培养基(Hybridoma，Gibico)大量培养四株杂交瘤细胞株。5天后收集培养上清，用Protein G亲和层析柱(美国Amersham公司)纯化抗体，测定浓度后分装，-70℃冻存。纯化结果见图4所示抗体纯度达到95％以上。
2.抗NSP1单克隆抗体CH001的功能活性鉴定
用酶联免疫吸附试验对实施例1的抗NSP1抗体和NSP1蛋白的结合进行鉴定。具体方法为：用包被液(0.1M碳酸盐缓冲液，pH9.6)稀释NSP1蛋白至2μg/mL包被ELISA 96孔板，每孔加入100μL，4℃过夜；PBST(PBS含0.5％Tween20)5％脱脂牛奶-洗涤缓冲液封闭，37℃孵育2h；PBST洗涤5次后，每个孔中加入100μL抗体(2μg/mL起始浓度，15个浓度梯度稀释)4℃过夜；以1：4000稀释的羊抗鼠二抗(北京中杉公司)100μL/孔加入到孔内，37℃孵育1h；过氧化物酶底物显色液100μL/孔，室温下15分钟后用2M硫酸中止反应，上机检测比色采用双波长450nm/690nm。
结果见图4显示：该纯化的单克隆抗体具有较高的抗体效价。
实施例2hm NSP1抗体的制备
经申请人前期鼠源单抗的检测，我们选择CH001杂交瘤细胞株制备人鼠嵌合抗体hm CH001。
1)抗体可变区基因片段的扩增及验证：
CH001杂交瘤细胞培养至对数生长期，Trizol-氯仿-异丙醇法抽提细胞总RNA；将干燥后的细胞总RNA溶于20μL水中，测定OD260/OD280，值为1.9。取14μL的RNA进行逆转录，以总RNA中的mRNA为模板，以OligodT15为引物，反转录扩增获得单链cDNA。
设计19条VH上游引物和17条Vκ上游引物，4条VH下游引物和3条Vκ下游引物引物：
Vκ5’上游引物：
Vκ-1
5’-GGG CCC AGG CGG CCG AGC TCG AYA TCC AGC TGA CTC AGC C-3’
Vκ-2
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCW CCC AGT C-3’
Vκ-3
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGM TMA CTC AGT C-3’
Vκ-4
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGY TRACAC AGT C-3’
Vκ-5
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TRA TGA CMC AGT C-3’
Vκ-6
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTM AGA TRA MCC AGT C-3’
Vκ-7
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTC AGA TGA YDC AGT C-3’
Vκ-8
5’-GGG CCC AGG CGG CCG AGC TCG AYA TYC AGA TGA CAC AGA C-3’
Vκ-9
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCA WCC AGT C-3’
Vκ-10
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG WGC TSA CCC AAT C-3’
Vκ-11
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTS TRA TGA CCC ART C-3’
Vκ-12
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTK TGA TGA CCC ARA C-3’
Vκ-13
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CBC AGK C-3’
Vκ-14
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TAA CYC AGG A-3’
Vκ-15
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CCC AGW T-3’
Vκ-16
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CAC AAC C-3’
Vκ-17
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTT TGC TGA CTC AGT C-3’
Vκ3’下游引物
VκR1
5’-AGA TGG TGC AGC CAC AGT TCG TTT KAT TTC CAG YTT GGT CCC-3’
VκR2
5’-AGA TGG TGC AGC CAC AGT TCG TTT TAT TTC CAA CTT TGT CCC-3’
VκR3
5’-AGA TGG TGC AGC CAC AGT TCG TTT CAG CTC CAG CTT GGT CCC-3’
VH 5’上游引物
VH 1
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR MAG CTT CAG GAG TC-3’
VH 2
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTB CAG CTB CAG CAG TC-3’
VH 3
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG CTG AAG SAS TC-3’
VH 4
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAR CTG CAA CAR TC-3’
VH 5
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAG CTB CAG CAR TC-3’
VH 6
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAR CTG CAG CAG TC-3’
VH 7
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAC GTG AAG CAG TC-3’
VH 8
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAS STG GTG GAA TC-3’
VH 9
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AWG YTG GTG GAG TC-3’
VH 10
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG SKG GTG GAG TC-3’
VH 11
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAM CTG GTG GAG TC-3’
VH 12
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG CTG ATG GAR TC-3’
VH 13
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAR CTT GTT GAG TC-3’
VH 14
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR AAG CTT CTC GAG TC-3’
VH 15
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAR STT GAG GAG TC-3’
VH 16
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTT ACT CTR AAA GWG TST G-3’
VH 17
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAA CTV CAG CAR CC-3’
VH 18
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAC TTG GAA GTG TC-3’
VH 19
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG GTC ATC GAG TC-3’
VH 3’下游引物
VH R1
5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC CGT GGT-3’
VH R2
5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC TGT GAG AGT GGT-3’
VH R3
5’-CGA TGG GCC CTT GGT GGA GGC TGC AGA GAC AGT GAC CAG AGT-3’
VH R4
5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC TGA GGT-3’
将Vκ5’上游引物、Vκ3’下游引物、VH 5’上游引物，VH 3’下游引物各溶为终浓度100pmol/μL，然后均以1：1体积比例混匀，分别扩增VH、VL基因，扩增条件为95℃4分钟，95℃30秒，60℃40秒，72℃45秒，30个循环；最后72℃延伸10分钟，电泳回收、纯化扩增的基因片段(见图5)，连至pMD-18T载体，转化大肠杆菌DH5α，测序后获得轻链、重链可变区序列。
2)PCR的引物的设计与基因扩增
重链扩增引物：
F：5’-GGTGTCCACTCGCTAGATGTGCAGCTGCAGGAATCGGGACCT-3’
R：5’-GCCCTTGGTGGATGCTGCAGAGACAGTGAC-3’
轻链扩增引物：
F：5’-ACAGACGCTCGCTGCCAAATTGTGCTCACTCAGTCTCCAG-3’
R：5’-TGCAGCCACCGTACGTTTGATTTCCAGTTTGGTC-3’
3)扩增hmCH001抗体重链、轻链
以上述重、轻链可变区连接到pMD18-T载体测序分析正确的为模板，分别以上述涉及的重链和轻链的上下游引物扩增人鼠嵌合抗体重链、轻链基因。
(1)PCR
反应体系：
反应条件：
(2)2％琼脂糖凝胶电泳，紫外下观察目的条带，切胶回收。
(3)胶回收试剂盒纯化目的DNA片段，去离子水洗脱。
5)双酶切IgG表达质粒
IgG表达质粒pFUSE-CHIg-hG1、pFUSE-CLIg-hk(购自Invivogen公司)包含IgG1型人源的重链和轻链(Kappa)恒定区碱基编码序列。
(1)pFUSE-CHIg-hG1、pFUSE-CLIg-hk模板载体的双酶切
反应体系：
反应条件：37℃酶切过夜。
(2)1％琼脂糖凝胶电泳，紫外下切胶回收。
(3)胶回收试剂盒纯化目的DNA片段，去离子水洗脱。
6)Infusion PCR重组表达质粒
反应体系：
反应条件：50℃孵育15min。
取5μL反应液转化感受态细菌，铺于相应抗性的平板上，次日挑克隆送测序。将测序结果正确的克隆保存菌种并扩大培养，提取质粒。
结果成功构建hmCH001的表达质粒。见图6。
7)hm CH001抗体的表达
(1)取250μL pFUSE-CHIg-hG1-hm CH001H(即50μg)于1mL的Opti-MEM培养基中，取250μL pFUSE-CLIg-hk-hm CH001K(即50μg)于1mL的Opti-MEM培养基中，取200μL的293Fectin于2.8mL的Opti-MEM培养基中，将上述三种混合液室温静置5min。
(2)然后将两个质粒混合液混合均匀后，补加500μL的Opti-MEM培养基混合均匀后直接加入转染试剂293Fectin的混合液，混合均匀后静置20min。期间处理293F细胞，将293F细胞离心后用293F Expression Medium重悬，然后计数以及用台盘蓝计算细胞活力比，吸取90×106个细胞于培养瓶中，用293F Expression Medium定容为94mL。
(3)20min结束后将6mL的DNA、293Fectin的复合物加入准备好的293F细胞中。
(4)将细胞放在摇床培养箱中培养，培养条件8％CO2，120rmp，37℃，6天后收集细胞上清。
8)hm CH001抗体的纯化
将收集的细胞上清用0.22μm的滤膜过滤，同时将平衡液和洗脱液过滤膜。用AKATA纯化仪按照Protein A纯化的标准步骤纯化，以1mL/min的速度上样，以1.5mL/min的速度洗脱。
结果成功表达并纯化hm F1-2D2抗体。SDS-PAGE见图7。
9)hm CH001纯化抗体酶联免疫吸附实验
用包被液(0.1M碳酸盐缓冲液，pH9.6)稀释F1蛋白至2μg/mL包被ELISA 96孔板，每孔加入100μL，4℃过夜；PBST(PBS含0.5％Tween20)5％脱脂牛奶-洗涤缓冲液封闭，37℃孵育2h；PBST洗涤5次后，每个孔中加入100μL F1-2D2(2μg/mL起始浓度，15个浓度梯度稀释)4℃过夜；以1：4000稀释的羊抗鼠二抗(北京中杉公司)100μL/孔加入到孔内，37℃孵育1h；过氧化物酶底物显色液100μL/孔，室温下15分钟后用2M硫酸中止反应，上机检测比色采用双波长450nm/690nm。
结果见图8示：该纯化的单克隆抗体hm CH001与NSP1蛋白有较好的结合活性，且能识别蛋白抗原的构象表位。
序列表
<110> 中国人民解放军东部战区疾病预防控制中心
<120> 人源化抗基孔肯雅病毒NSP1抗体及其应用
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gacattgtga tgacccagtc tcacaaagtc atgtccacat cagtaggaga cagggtcagc 60
atcacctgca aggccagtca gattagtgac gtggctgtag cctggcatca acagaaacca 120
ggacaatctc ctaaaccact gatttactcg gcatcctacc agtacactgg agtccctgat 180
cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 240
gaagacctgg cagtttatta ctgtcagccg attcaagtgt acgactggac gttcggtgga 300
ggcaccaagc tggaaatcaa a 321
<210> 2
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Ile Ser Asp Val Ala
20 25 30
Val Ala Trp His Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Gln Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Pro Ile Gln Val Tyr Asp Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 3
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Ile Ser Asp Val Ala
1 5
<210> 4
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ser Ala Ser
1
<210> 5
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gln Pro Ile Gln Val Tyr Asp Trp Thr
1 5
<210> 6
<211> 354
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc ctggaggatc cctgaaactc 60
tcctgtgcag cctcagggaa ctgagattct tgtagatgga tgagttgggt ccggcaggct 120
ccagggaaag ggctagaatg gattggagaa attaatccag atagcagtaa gataaactat 180
atgccatctc taaaggataa attcatcatc tccagagaca acgccaaaaa tacgctgtac 240
ctgcaaatga gcaaagtgag atctgaggac acagcccttt attactgtgc aagaggtcct 300
cggtactaca aggaggactt ctggggtcaa ggaacctcag tcaccgtctc ctca 354
<210> 7
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Glu Asp Leu Ser Arg
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Asp Ser Ser Lys Ile Asn Tyr Met Pro Ser Leu
50 55 60
Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Arg Tyr Tyr Lys Glu Asp Phe Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 8
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gly Tyr Glu Asp Leu Ser Arg Trp
1 5
<210> 9
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ile Asn Pro Asp Ser Ser Lys Ile
1 5
<210> 10
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Ala Arg Gly Pro Arg Tyr Tyr Lys Glu Asp Phe
1 5 10
<210> 11
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tagagctagc gaattcgaat tccaccatgg agaccgatac c 41
<210> 12
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ttgcggccgc ggatccgcgg ccgcttatca cttgc 35
<210> 13
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gggcccaggc ggccgagctc gayatccagc tgactcagcc 40
<210> 14
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gggcccaggc ggccgagctc gayattgttc tcwcccagtc 40
<210> 15
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gggcccaggc ggccgagctc gayattgtgm tmactcagtc 40
<210> 16
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gggcccaggc ggccgagctc gayattgtgy tracacagtc 40
<210> 17
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gggcccaggc ggccgagctc gayattgtra tgacmcagtc 40
<210> 18
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gggcccaggc ggccgagctc gayattmaga tramccagtc 40
<210> 19
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gggcccaggc ggccgagctc gayattcaga tgaydcagtc 40
<210> 20
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gggcccaggc ggccgagctc gayatycaga tgacacagac 40
<210> 21
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gggcccaggc ggccgagctc gayattgttc tcawccagtc 40
<210> 22
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gggcccaggc ggccgagctc gayattgwgc tsacccaatc 40
<210> 23
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gggcccaggc ggccgagctc gayattstra tgacccartc 40
<210> 24
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gggcccaggc ggccgagctc gayattktga tgacccarac 40
<210> 25
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gggcccaggc ggccgagctc gayattgtga tgacbcagkc 40
<210> 26
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gggcccaggc ggccgagctc gayattgtga taacycagga 40
<210> 27
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gggcccaggc ggccgagctc gayattgtga tgacccagwt 40
<210> 28
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gggcccaggc ggccgagctc gayattgtga tgacacaacc 40
<210> 29
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gggcccaggc ggccgagctc gayattttgc tgactcagtc 40
<210> 30
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
agatggtgca gccacagttc gtttkatttc cagyttggtc cc 42
<210> 31
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
agatggtgca gccacagttc gttttatttc caactttgtc cc 42
<210> 32
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
agatggtgca gccacagttc gtttcagctc cagcttggtc cc 42
<210> 33
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gctgcccaac cagccatggc cctcgaggtr magcttcagg agtc 44
<210> 34
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gctgcccaac cagccatggc cctcgaggtb cagctbcagc agtc 44
<210> 35
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gctgcccaac cagccatggc cctcgaggtg cagctgaags astc 44
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<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gctgcccaac cagccatggc cctcgaggtc carctgcaac artc 44
<210> 37
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gctgcccaac cagccatggc cctcgaggty cagctbcagc artc 44
<210> 38
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gctgcccaac cagccatggc cctcgaggty carctgcagc agtc 44
<210> 39
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gctgcccaac cagccatggc cctcgaggtc cacgtgaagc agtc 44
<210> 40
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gctgcccaac cagccatggc cctcgaggtg aasstggtgg aatc 44
<210> 41
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
gctgcccaac cagccatggc cctcgaggtg awgytggtgg agtc 44
<210> 42
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
gctgcccaac cagccatggc cctcgaggtg cagskggtgg agtc 44
<210> 43
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gctgcccaac cagccatggc cctcgaggtg camctggtgg agtc 44
<210> 44
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
gctgcccaac cagccatggc cctcgaggtg aagctgatgg artc 44
<210> 45
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gctgcccaac cagccatggc cctcgaggtg carcttgttg agtc 44
<210> 46
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
gctgcccaac cagccatggc cctcgaggtr aagcttctcg agtc 44
<210> 47
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
gctgcccaac cagccatggc cctcgaggtg aarsttgagg agtc 44
<210> 48
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
gctgcccaac cagccatggc cctcgaggtt actctraaag wgtstg 46
<210> 49
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
gctgcccaac cagccatggc cctcgaggtc caactvcagc arcc 44
<210> 50
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
gctgcccaac cagccatggc cctcgaggtg aacttggaag tgtc 44
<210> 51
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
gctgcccaac cagccatggc cctcgaggtg aaggtcatcg agtc 44
<210> 52
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
cgatgggccc ttggtggagg ctgaggagac ggtgaccgtg gt 42
<210> 53
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
cgatgggccc ttggtggagg ctgaggagac tgtgagagtg gt 42
<210> 54
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
cgatgggccc ttggtggagg ctgcagagac agtgaccaga gt 42
<210> 55
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
cgatgggccc ttggtggagg ctgaggagac ggtgactgag gt 42
<210> 56
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
ggtgtccact cgctagatgt gcagctgcag gaatcgggac ct 42
<210> 57
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
gcccttggtg gatgctgcag agacagtgac 30
<210> 58
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
acagacgctc gctgccaaat tgtgctcact cagtctccag 40
<210> 59
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
tgcagccacc gtacgtttga tttccagttt ggtc 34
Claims (5)
1.一种含有抗NSP1单克隆抗体，其特征在于，所述单克隆抗体的重链可变区具有如SEQID NO:7所示的氨基酸序列，轻链可变区具有如SEQ ID NO:2所示的氨基酸序列。
2.权利要求1所述含有抗NSP1单克隆抗体，其特征在于，所述重链可变区具有如SEQ IDNO:6所示DNA序列编码的氨基酸序列，轻链可变区具有如SEQ ID NO:1所示DNA序列编码的氨基酸序列。
3.一种含有单克隆抗体重链或轻链的核苷酸编码的表达载体，其特征在于含有SEQ IDNO:6所示的重链可变区DNA序列和SEQ ID NO:1所示的轻链可变区DNA序列。
4.根据权利要求3所述的表达载体，其特征在于，所述载体为抗体IgG1亚型分泌型真核表达载体。
5. 一种含有权利要求4所述表达载体的宿主细胞，宿主细胞为FreeStyle™ 293-Fcells。
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