CN102229671B - 抗IMMT全人源Fab抗体及其在制备抗肝癌药物中的应用 - Google Patents
抗IMMT全人源Fab抗体及其在制备抗肝癌药物中的应用 Download PDFInfo
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Abstract
抗IMMT全人源Fab抗体及其在制备抗肝癌药物中的应用,轻链的氨基酸序列如SEQIDNo:1所示,重链的氨基酸序列如SEQIDNo:2所示;轻链的核苷酸序列如SEQIDNo:3所示,重链的核苷酸序列如SEQIDNo:4所示;体外观察hFabr1抗肝癌效应结果表明,hFabr1能抑制HepG2细胞的增殖和侵袭,诱导HepG2细胞凋亡。动物体内实验表明hFabr1能明显抑制荷瘤裸鼠体内HepG2移植瘤的生长。全人源抗IMMT基因工程抗体的获得不仅为原发性肝癌的预防和治疗带来了希望,也为研制能用于肝癌和其它恶性肿瘤生物治疗的抗体药物提供了新的思路和技术贮备。
Description
技术领域
本发明属于生物工程领域,涉及一种抗IMMT全人源Fab抗体及其在制备抗肝癌细胞HepG2药物中应用。
背景技术
原发性肝癌是危害人类健康的重要疾病之一,多次被我国政府列为研究和防治的重点。肝癌的治疗长期以来一直是一个世界性的难题。肝癌发病隐匿,患者就诊时多属中、晚期,而且肝癌是一种恶性程度高、浸润和转移性强以及先天性耐药的恶性肿瘤,手术、放疗、化疗及介入等常规治疗疗效均不理想,也不能从根本上解决肝癌的转移和复发,特别是面对那些分化程度低或未分化、已经远处转移、病期较晚的肝癌患者可谓束手无策。此外抗癌药物的普通制剂给药后在全身分布,杀伤癌细胞的同时也损伤正常细胞,产生严重的不良反应。因此研发特异性强、靶向性强、疗效高、副作用小的药物成为一项关系到人民生命健康的重要工程,已成为肿瘤领域的研究热点。
南京医科大学卫生部抗体技术重点实验室承担卫生部科学研究基金课题“肝癌早期相关基因和肿瘤标志物筛选”的研究课题期间,使用经典方法二乙基亚硝胺诱导Wistar大鼠成功建立肝癌模型,分别把不同时期(非特异性肝损伤、肝硬化、肝细胞不典型增生、早期肝癌结节和肝癌晚期出现远处转移灶)的肝脏组织提取RNA,进行Affymetrix芯片检测(GeneChipRat Genome 2302.0Array)。分析发现线粒体内膜蛋白IMMT(Inner Membrane Protein,Mitochondrial)在大鼠肝癌呈高水平表达,是正常组织的25倍,而在非特异损伤和硬化阶段表达却没有检测到表达升高,并经过荧光定量PCR技术和绿色荧光蛋白示踪技术所证实。大鼠肝癌发生与人体肝癌发生的过程非常相似,而大鼠IMMT与人体的IMMT高度同源(100%)。本实验室已经在临床原发性肝癌组织标本和相应的非肝癌肝组织标本中通过荧光实时定量PCR比较发现,IMMT表达水平在肝癌组织中高于非肝癌肝组织,IMMT可能通过影响肝癌细胞内骨架蛋白的构像和分布,从而改变肝癌细胞的运动能力,参与肝癌侵袭机制。IMMT高表达的肝癌细胞获得淋巴细胞的“伪装”,逃避人体免疫系统的识别和杀伤,更易进入淋巴结,形成转移。因此IMMT成为肝癌靶向治疗的一个良好的靶点。通过在美国Pubmed和中国CNKI数据库的检索,目前尚未见相关研究报道。目前抗肝癌IMMT鼠源单克隆抗体在本实验室已研制成功。用于肝癌治疗的人源抗IMMT基因工程抗体,目前国内外均无报道。
发明内容
技术问题:本发明旨在提供抗IMMT Fab抗体重、轻链可变区基因及其编码的多肽,以及该抗体在制备抗肝癌药物中的应用。
技术方案:抗IMMT全人源Fab抗体,轻链的氨基酸序列如SEQ ID No:1所示,重链的氨基酸序列如SEQ ID No:2所示。
抗IMMT全人源Fab抗体,轻链的核苷酸序列如SEQ ID No:3所示,重链的核苷酸序列如SEQ ID No:4所示。
所述的抗IMMT全人源Fab抗体在制备抗肝癌药物中的应用。
本发明人从原发性肝癌患者全血中分离的B淋巴细胞中克隆了基因组所有的抗体重、轻链可变区基因,采取基因工程方法,构建库容量为1.3×109的全人源免疫型Fab抗体库。用纯化的IMMT蛋白对噬菌体抗体库进行富集筛选,分离了1株抗IMMT型的特异性人源Fab抗体。测序结果证实其Fd链和L链的DNA序列及氨基酸序列(命名为hFabr1)。对克隆可溶性表达后的沉淀及上清进行SDS-PAGE和Western blot分析,证实在约26kD和30kD左右有二条目的条带,确定抗体有可溶性表达。ELISA显示克隆与IMMT蛋白反应阳性(OD450nm=1.218),说明该克隆是与IMMT有特异性结合活性的Fab抗体片段。
有益效果:体外观察hFabr1抗肝癌效应结果表明,hFabr1能抑制HepG2细胞的增殖和侵袭,诱导HepG2细胞凋亡。动物体内实验表明hFabr1能明显抑制荷瘤裸鼠体内HepG2移植瘤的生长。全人源抗IMMT基因工程抗体的获得不仅为原发性肝癌的预防和治疗带来了希望,也为研制能用于肝癌和其它恶性肿瘤生物治疗的抗体药物提供了新的思路和技术贮备。
附图说明
图1为抗体重链、轻链可变区基因及其抗体hFabr1基因PCR产物,其中M:DNA Marker;1:Fab;2:Lκ;3:Lλ;4:Fd;5:CL;6:CH;7:Vκ;8:Vλ;9:VH;
图2为SDS-PAGE检测纯化的hFabr1抗体;
图3为hFab hFabr1作用48h后HepG2细胞凋亡的分布;
图4为LSCM检测细胞凋亡(48h晚期凋亡细胞),其中A.空白对照组:正常细胞膜联蛋白V(-)PI(-);B.低剂量组:晚期凋亡细胞膜联蛋白V(+)PI(+);C.中剂量组:晚期凋亡细胞膜联蛋白V(+)PI(+);D.高剂量组:晚期凋亡细胞膜联蛋白V(+)PI(+);
图5为透射电镜观察HepG2细胞超微结构,其中A.空白对照组:细胞结构清楚,核膜完整,核内染色质分布均匀。胞质内可见较多的结构清晰的线粒体等细胞器;B.低剂量组:细胞核内染色质趋边凝集,胞质内线粒体轻度肿胀;C.中剂量组:细胞核内染色质趋边凝集或成块状凝集。胞质内线粒体肿胀、空化,胞质轻度空化;D.高剂量组:细胞核呈固缩状,核内异染色质趋边凝集,胞质电子密度加深,细胞质空化明显。
图6为Transwell小室HepG2细胞迁徙实验,其中A.空白对照组;B.低剂量组;C.中剂量组;D.高剂量组;
具体实施方式
1.大容量全人源免疫型抗IMMT Fab噬菌体抗体库的构建
从原发性肝癌患者全血中分离的B淋巴细胞中克隆了基因组所有的抗体重、轻链可变区基因,采取基因工程方法,构建库容量为1.3×109的全人源免疫型Fab抗体库。
2.抗IMMT Fab抗体的筛选、表达、纯化及鉴定
用纯化的IMMT蛋白对构建的噬菌体抗体库进行富集筛选,分离了1株抗IMMT的特异性人源Fab抗体。
SDS-PAGE与Western-blot分析表明,转化工程菌能正确表达Fab抗体。变性条件下,Fab抗体分子的异二聚体分解为Fd与Kappa链。
经Protein L纯化的hFabr1基本与其他蛋白分离,SDS/PAGE进一步证实了其结构与分子量与Fab相符,同时也证实了其纯度。
ELISA鉴定结果显示纯化的hFabr1有明显的与IMMT结合的能力,随剂量的递减,OD值呈下降趋势。
3.免疫荧光检测Fab抗体与IMMT结合位点
hFabh1与肝癌HepG2细胞表面的IMMT结合,结合部位为细胞膜。
4.抗体在预防及治疗肝癌试验中的应用
主要包括体外细胞试验和体内裸鼠模型试验
4.1hFabr1抗体对肝癌HepG2细胞增殖、侵袭及凋亡的作用研究
hFabr1抗体对肝癌HepG2细胞增殖和侵袭有抑制作用,并能诱导HepG2细胞凋亡。
4.2hFabr1抗体对裸鼠肝癌HepG2细胞移植瘤的作用研究
高剂量组肿瘤生长速度减慢,中剂量组同样减慢但比高剂量组快,阴性对照组与空白对照组差异不明显;hFab r1二个剂量组肿瘤生长抑制率比对照组高,在两周内均呈上升趋势,但高剂量组上升趋势更大。说明hFab r1对肿瘤的生长有抑制作用,且抑制效果与用药剂量及用药时间关系密切,但并不能完全停止肿瘤的生长或使肿瘤体积缩小。
实施例1全人源免疫型Fab噬菌体抗体库的构建
采集原发性肝癌患者全血80份,分离淋巴细胞,采用Invitrogen公司的Trizol抽提总RNA,用oligo(dT)20为引物,逆转录生成cDNA。然后利用特异性引物PCR分别扩增其重、轻链可变区基因。经Overlap PCR合成Fab基因(图1),与经相同酶切的表达载体pComb3XSS连接,构建Fab的原核重组表达载体;电转化感受态大肠杆菌XL1-Blue,构建了容量为1.3×109的全人源免疫型Fab抗体库。
所述构建的Fab基因与pComb3xSS载体连接是指:将纯化定量后的Fab基因分别以SfiI内切酶进行双酶切消化;纯化、定量后与同样双酶切pComb3xSS载体连接;所述的转化是指:a)0.2cm电转杯,25μF,2.5kV,200Ω的电转条件转化感受态大肠杆菌XL1-Blue;b)转化产物加入LB培养基后37℃振荡培养2h,10倍梯度稀释将菌液涂布于SOBAG琼脂板上,30℃过夜培养;c)次日计算平板上的克隆数,估算库容为1.3×109;d)将电转化后菌液加入辅助噬菌体M13K07超感染,离心沉淀菌体,用LB培养基重悬,37℃振荡过夜,离心沉淀菌体,吸取上清即为Fab噬菌体表面展示文库。
实施例2抗IMMT特异性抗体的筛选、表达及纯化
(1)用纯化的IMMT的包被固相筛选ELISA板,每孔1μg,洗涤,加封闭液,洗涤,加入噬菌体抗体库抗体,洗涤去除未结合的噬菌体抗体;加入胰蛋白酶,洗脱特异性结合的噬菌体抗体,感染增值,辅助噬菌体M13K07超感染;重复以上筛选步骤,共进行五轮“吸附-洗脱-扩增”富集筛选;
(2)将最后一轮筛选且增值得到的噬菌体稀释后铺于培养板上培养过夜,挑取31个单菌落于细胞培养板中,振摇培养过夜;从第一块板各孔中分别转移5μL菌液至第二块板,振摇培养;加辅助噬菌体M13K07超感染,振摇培养;离心,培养基重悬沉淀,振摇培养过夜。离心取上清进行ELISA检测,测定每孔450nm和650nm吸光值,按A450nm-A650nm计算每孔吸光值;当P/N值(Positive/Negative)大于2.1时,该菌株为阳性单克隆噬菌体菌株;共得到25个阳性克隆。阳性克隆经核酸序列分析后,发现有9株不同序列Fab克隆与人IgG-Fc重组蛋白有较强的结合活性,Fab重、轻链可变区基因序列分析应用以下两个服务器:
http://imgt.cines.fr/IMGT_vquest/vquest?livret=0&Option=mouseIg
http://blast.ncbi.nlm.nih.gov/Blast.cgi
对所得1条序列与现有已报道的其他抗体基因进行同源性比较,并分析其胚系基因来源,结果如下:
hFabr1抗体的重链可变区基因的核苷酸序列:
Fd
ATGGCCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGTAGCAGCTCGCTACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCCCCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTAGTGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAG
hFabr1抗体的重链可变区基因的氨基酸序列:
Fd
MAQVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGVAARYWYFDLWGRGTLVTVSPASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAGQHHHHHHGAYPYDVPDYAS
hFabr1抗体的轻链可变区基因的核苷酸序列:
L
GAGCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGTAACTTACTACTGTCAACAGAGTTACAGTACCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
hFabr1抗体的轻链可变区基因的氨基酸序列:
L
ELQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFVTYYCQQSYSTPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRGEC
(3)将1株阳性克隆噬菌体感染非抑制型Top10F’,含有重组质粒pComb3x-Fab的菌株接种10mL的LB液体培养基中(含有100ug/mL的氨苄青霉素),37℃摇床培养至OD600nm至0.9,加入终浓度为1mM的IPTG,37℃诱导4小时。用SDS-PAGE和Western-blot鉴定。
(4)Fab单链抗体的纯化
缓冲液
结合缓冲液:20mM磷酸钠缓冲液,0.5M NaCl,45mM咪唑,pH7.4.
洗脱缓冲液:20mM磷酸钠缓冲液,0.5M NaCl,500mM咪唑,pH7.4.
样品准备
经鉴定的高表达菌株,挑单克隆置25mL的LB液体培养基中(含有100ug/mL的氨苄青霉素),250rpm,37℃培养过夜。
把25mL过夜培养物加至500mL的LB液体培养基中(含有氨苄青霉素),250rpm,37℃培养2小时,加入IPTG至终浓度为1mm。再诱导表达4小时。
培养物5,000g离心20分钟,弃上清。用50mL预冷的结合缓冲液(含有1mM的蛋白酶抑制剂PMSF)重悬菌体沉淀,在冰浴条件下超声波破碎细菌。
在菌体裂解物中加入终浓度为1%的Triton X-100,室温轻轻搅拌30分钟,以利于破碎包涵体。20,000g离心30分钟,上清液过0.45μm滤膜,备用。
纯化
把NiSO4亲和层析柱安装至蛋白质纯化仪上,用10个柱床体积的结合缓冲液冲洗酒精。用10个柱床体积的结合缓冲液平衡柱床后,上样品,流速为1mL/分钟。
用结合缓冲液洗涤柱床至A280nm吸光度到基线,用5-10个柱床体积的洗脱缓冲液洗脱目的蛋白,用SDS-PAGE鉴定(图2)。
实施例3具有结合活性的全人源Fab抗体的鉴定
用于实验的肝癌HepG2细胞系购自中国科学院上海细胞研究所细胞库。
与IMMT结合特性分析,ELISA鉴定结果显示纯化的hFabr1有明显与IMMT结合的能力,且随hFabr1剂量的递减,OD值呈下降趋势。免疫荧光检查结果显示hFabr1与肝癌HepG2细胞表面的IMMT结合,结合部位为细胞膜。
ELISA鉴定hFabr1与IMMT的结合能力(OD450nm)
实施例4hFabr1抗体对肝癌HepG2细胞增殖、侵袭及凋亡的作用
实验分空白对照组、低剂量租、中剂量组和高剂量组四组。MTT检测不同组别在不同时间点的细胞OD570nm值,显示同一时间点不同剂量组与空白对照组比较OD值显著降低,随hFabr1剂量增加OD值出现降低趋势。hFabr1对HepG2细胞生长有抑制作用,随剂量增加和作用时间延长,细胞增殖速度减慢,高剂量组对HepG2细胞生长抑制作用最明显。
FCM检测hFabr1对HepG2细胞周期的影响,hFabr1作用HepG2细胞24h后各剂量组与对照组比较,G0/G1期细胞均明显增多(P<0.05)。随剂量增加G0/G1期细胞呈增多趋势,S期细胞均明显降低(P<0.05),随剂量增加S期细胞呈降低趋势,G2/M期细胞均降低(P<0.05),随剂量增加G2/M期细胞亦呈降低趋势。
细胞凋亡检测各剂量组HepG2细胞早期凋亡率与空白对照组比较均增加,差异均具有统计学意义(P<0.05);并随hFabr1剂量增加,早期凋亡率亦增加,与hFabr1剂量大小呈正相关(r=0.641,P<0.05)。晚期凋亡率逐渐增多,与空白对照组比较差异均有统计学意义(P<0.05);与空白对照组比较,hFabr1不同剂量组的正常细胞百分比均下降,差异均具有统计学意义(P<0.05);坏死细胞百分比均下降,差异均具有统计学意义(P<0.05)(图3)。
Transwell小室HepG2细胞迁徙实验,低剂量组与空白对照组比较无差异(P>0.05),迁徙的HepG2细胞中剂量组与低剂量组比较有显著差异(P<0.01),高剂量组与中剂量组比较有差异(P<0.05),高剂量组在相同时间通过微孔滤膜的数量较其他各组显著减少(图6)。
实施例5hFabr1抗体对裸鼠肝癌HepG2细胞移植瘤的作用研究
实验裸鼠分空白对照组(DMEM)、低剂量组(0.8mg/mL)、高剂量组(1.6mg/mL)和阴性对照组(抗NP11-4Fab1.6mg/mL)。
治疗第10d、15d、20d时各组瘤体大小有区别(P<0.05),对照组肿瘤生长速度较快,治疗组生长速度较慢。随着时间推移高剂量组和低剂量组肿瘤生长速度减慢,治疗第20天时高剂量组肿瘤生长抑制作用最明显。空白对照组与阴性对照组的肿瘤湿重无显著差异(P﹥0.05),各剂量组肿瘤湿重低于对照组(P<0.01)。高剂量组抑瘤率高于低剂量组(P<0.05)。
SEQUENCE LISTING
<110> 南京医科大学
<120> 抗IMMT 全人源Fab抗体及其在制备抗肝癌药物中的应用
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 214
<212> PRT
<213> 人工序列
<400> 1
Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Val Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Leu Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 2
<211> 252
<212> PRT
<213> 人工序列
<400> 2
Met Ala Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30
Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
35 40 45
Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Gly Val Ala Ala Arg Tyr Trp Tyr Phe Asp Leu Trp
100 105 110
Gly Arg Gly Thr Leu Val Thr Val Ser Pro Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr Ser Gly Gln Ala Gly Gln His His His His His His
225 230 235 240
Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser
245 250
<210> 3
<211> 645
<212> DNA
<213> 人工序列
<400> 3
gagctccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg taacttacta ctgtcaacag agttacagta ccccattcac tttcggccct 300
gggaccaaag tggatatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagcttgc ccgtcacaaa gagcttcaac aggggagagt gttag 645
<210> 4
<211> 759
<212> DNA
<213> 人工序列
<400> 4
atggcccagg tgcagctggt gcagtctggg ggaggcctgg tcaagcctgg ggggtccctg 60
agactctcct gtgcagcctc tggattcacc ttcagtagct atagcatgaa ctgggtccgc 120
caggctccag ggaaggggct ggagtgggtc tcatccatta gtagtagtag tagttacata 180
tactacgcag actcagtgaa gggccgattc accatctcca gagacaacgc caagaactca 240
ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ctgtgtatta ctgtgcgaga 300
ggggtagcag ctcgctactg gtacttcgat ctctggggcc gtggcaccct ggtcaccgtc 360
tcccctgcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420
tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480
gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540
tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660
gagcccaaat cttgtgacaa aactagtggc caggccggcc agcaccatca ccatcaccat 720
ggcgcatacc cgtacgacgt tccggactac gcttcttag 759
Claims (3)
1.抗IMMT 全人源Fab抗体,其特征在于轻链的氨基酸序列如SEQ ID No:1所示,重链的氨基酸序列如SEQ ID No:2所示。
2.表达抗IMMT 全人源Fab抗体的核苷酸,其特征在于轻链的核苷酸序列如SEQ ID No:3所示,重链的核苷酸序列如SEQ ID No:4所示。
3.权利要求1所述的抗IMMT 全人源Fab抗体在制备抗肝癌药物中的应用。
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WO2006064136A1 (fr) * | 2004-12-16 | 2006-06-22 | Centre National De La Recherche Scientifique (Cnrs) | Production de formats d'anticorps et applications immunologiques de ces formats |
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CN101565465A (zh) * | 2008-05-14 | 2009-10-28 | 复旦大学 | 一种抗刚地弓形虫表面抗原1人源抗体Fab片段及其编码基因 |
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