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抗Aβ人源单链抗体基因的筛选和全抗体基因的构建及表达研究
【作者】 贾静；
【导师】 梁平；
【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2008， 硕士
【摘要】 阿尔茨海默病（Alzheimer disease,AD）是一种以老年人中进行性记忆障碍和智能衰退为主要临床特征的中枢神经系统退行性疾病。老年斑（senile plaque,SP）、神经元纤维缠结（neurofibrillary tangles,NFT）和神经突触减少或神经元丢失是AD的主要病理特征。其中老年斑的主要成分是β-淀粉样多肽（beta-amyloidpeptide,Aβ）聚集形成的纤维,而Aβ纤维被认为是导致神经元损伤及认知功能衰退的主要致病物质。针对Aβ多肽在发病中的关键作用,研究如何阻止Aβ多肽聚集及清除大脑病变区已形成的Aβ多肽纤维已成为研究AD治疗的前沿领域,抗Aβ多肽抗体治疗AD也成为AD研究中的一个热点。动物试验已证明抗Aβ多肽抗体有清除小鼠大脑中Aβ纤维斑块防治AD的作用,但至今尚未见人源化抗体有临床应用的报道。本课题研究从人源噬菌体单链抗体库中筛选针对Aβ的单链抗体可变区（scFv）基因,并以基因工程构建人源抗Aβ全抗体的真核表达体系。主要包括两部分:第一,从构建的人源噬菌体单链抗体库中筛选抗Aβ的scFv基因,在原核细胞中进行可溶性表达,并进行抗体结合活性和生物学活性检测;第二,重组IgG全抗体轻重链基因及全抗体单链基因,构建真核表达体系,进行真核细胞表达的初步研究。一、抗Aβ人源scFv基因的筛选、原核表达与鉴定以Aβ1-42多肽为抗原对抗体库进行4轮富集筛选:用辅助噬菌体M13K07感染E.coli TG1转化菌,展示出噬菌体形式的抗体库。将此抗体库加入到用Aβ1-42多肽包被的96孔板内,孵育一段时间后洗涤,能够与抗原特异结合的scFv噬菌体克隆将被保留在96孔板内壁,然后用pH 2.2的甘氨酸-HCl缓冲液将特异结合的噬菌体洗脱下来,经pH8.0的Tris-HCl中和后,感染处于对数生长期的E.coli TG1,以扩增抗原特异性噬菌体克隆。这样,经过4轮“吸附-洗脱-扩增”的富集过程,抗体库中与Aβ1-42多肽特异性结合的噬菌体克隆从第1轮的3.31×10-4增加到第4轮的7.65×10-2,得到了高度富集,多克隆噬菌体Elisa检测也显示了明确的富集效果。随机挑取80个克隆,用M13K07感染后使scFv展示于噬菌体颗粒表面,ELISA筛选展示有抗Aβ1-42多肽scFv的噬菌体克隆。检测结果显示,得到7个阳性克隆,其中4个克隆ELISA检测A值高于阴性对照5倍以上,另外3个在2.5～3倍之间。所有阳性克隆均与无关抗原BSA无交叉反应。对其中A值最高的2个克隆（A10、F2）的噬菌体颗粒上清感染E.coli HB2151,30℃,IPTG诱导表达20h,分别制备胞周质、培养基上清和全细胞提取物。经12%SDS-PAGE和western blot分析,目的蛋白相对分子量为33kD,主要集中于全细胞提取物,表达量占全菌蛋白60%左右。表达抗体ELISA检测上清中和全菌蛋白中A值分别高于阴性对照5倍以上,在胞周质中高于2倍以上。抗体与Aβ1-40也有结合反应,但与BSA无交叉反应,显示scFv抗体对Aβ具有特异性,结合位点主要在Aβ的N端。表达抗体的生物学活性鉴定示,可与人脑病变组织内淀粉样斑块中的抗原物质Aβ纤维结合,证明表达的scFv抗体可以应用于体内生物学效应实验。将A10、F2克隆菌测序,用blast分析得到的scFv基因序列,从中分别确定了VH与VL的DNA序列,推导得到的氨基酸序列具有典型的抗体可变区结构。2个克隆的基因序列一致,表明2个克隆菌可能来源于同一株原始噬菌粒。二、抗Aβ人源全抗体基因的重组构建、真核表达研究首先对前期获得的A10克隆scFv基因进行NCBI blast比对,获得VH和VL基因相应的成熟信号肽基因,并合成信号肽基因,再设计特异引物分别扩增A10克隆的VH、VL、scFv基因和人源IgG的CH、CL、FC段基因,并利用重组PCR（overlap PCR）方法重组IgG全抗体重链基因（H信号肽基因+VH基因+CH基因）、IgG全抗体轻链基因（L信号肽基因+VL基因+CL基因）和全抗体单链基因（H信号肽基因+scFv基因+FC段基因）,然后分别克隆到pcDNA3.1真核表达载体内。菌液PCR、双酶切鉴定和DNA测序结果均显示,重组的三种基因片段大小与预期一致,基因序列与预期的序列完全一致,读码框架和插入真核表达载体的方向完全正确。将IgG全抗体重链基因-载体质粒和IgG全抗体轻链基因-载体质粒共转染CHO细胞,表达IgG全抗体;将全抗体单链基因-载体质粒转染CHO细胞,表达单链全抗体（scFv抗体+FC段）。两种转染细胞初步的表达产物ELISA结果未见表达上清与Aβ1-42有阳性反应,后续工作仍需优化细胞表达条件。现有的工作进展为今后进一步优化表达、动物试验及应用研究奠定基础。目前,虽然国内外已有几家筛选到人源抗Aβ抗体的报道,但都仍处于实验室研究阶段,也未见全抗体或进入临床试验的报道。我们所得到的抗Aβ序列经过比对,与现有已发表的抗Aβ抗体序列均不同,表明是一个新的抗Aβ抗体。现已在国内申报了专利,同时已完成了全抗体真核表达体系构建工作,后续的优化表达工作正在进行。本课题研究工作的结果有利于今后进一步开展对AD的免疫治疗和临床应用研究。
【Abstract】 Alzheimer’s disease（AD） is a degenerative disease of the central nervous system, progressive dysmnesia and intelligent recession as main clinical feature. It is neuropathologically characterized by the deposition of extracellularβ-amyloid （Aβ）-containing plaques, intracellular neurofibrillary tangles, reduced synaptic density and neuronal loss in selected brain areas. The aggregation of beta-amyloid （Aβ） peptides in the senile plaques is believed to contribute to neuronal dysfunction and death, and impairment of memory in AD patients. Research on preventing Aβassembly, reducing parenchymal plaque burden, decreasing total Aβbrain levels and reversing cognitive deficits has become valuable. Anti-Aβantibody studying provide a hopeful approach for AD therapy. In animal trials, direct administration of anti-Aβantibodies has proved successful in clearing amyloid from the brain and reversing memory deficits. But the report of successful clinical use of anti-Aβhumanized antibodies to stimulate Aβclearance hasn’t been found.Our study is dedicated to preparing humanized scFv antibody agaist Aβ1-42 by phage display technic and constructing entire antibody gene. Two parts is included: first, to screen out the specific antibody clones againstβamyloid peptide 1-42 from human phage-display single-chain Fv antibody library, clone the antibody gene and express it in a bacterial system, and confirm its antigen-binding ability and biological activity. Second, to construct IgG entire antibody gene and entire scFv gene, and express them in a eukaryotic system.1. Screening, bacterial expression and characterization of human scFv antibody against Aβ1-42 peptide.βamyloid peptide 1-42 was bound on the solid surface of 96 wells plate as antigen to screen the binding clones from a human phage-display scFv antibody library. After four rounds of "binding-elution- amplification", a large enrichment was observed. 80 picked randomly well-separated colonies were identified by ELISA test. The results showed that 7 positive colonies had remarkable binding abilities with highest OD490 value and none of cross-reactivity with other kinds of antigen. The specific positive phage clones were infected into the host E. coli HB2151 to express soluble scFv antibodies and three different kinds of extract （culture medium, periplasmic space and whole cell extract） were prepared after IPTG induction. The soluble scFv antibody from clone A10 was expressed successfully and identified by SDS-PAGE and western blot, and antigen-binding activities were determined by ELISA. The results of SDS-PAGE and western blot showed a new protein band with a proximate MW of 33KD was mainly in whole cell extract. The value of the expression products was five folds as that of control by OD 490nm test, and none of cross-reactivity with BSA. The results of biological activity showed the scFv antibody could bind to Aβfibrella in senior plaques of AD patients’ brains. The positive scFv antibodies gene was sequenced. DNA sequencing data demonstrated that the gene of the positive antibody specifically against Aβ1-42 was the scFv gene and the deduced amino acids sequence confirmed its typical antibody V domain structure. 2. Construction and eukaryotic expression of entire antibody gene.At first, we synthesized H and L signal peptide gene obtained from NCBI blast results. Then we amplified the target antibody gene by using specific primers. We acquired IgG entire antibody H gene（H signal peptide gene +VH gene +CH gene）, IgG entire antibody L gene （L signal peptide gene +VL gene +CL gene） and entire scFv gene （H signal peptide gene +scFv gene +FC gene） by performing overlap PCR, then cloned the gene into pcDNA3.1 vector respectively. The results of PCR and DNA sequencing data showed three kinds of constructed gene confirmed their expectant DNA sequence. IgG entire antibody H gene-vector plasmid and IgG entire antibody L gene-vector plasmid were transferred to CHO cell together with the purpose to produce IgG entire antibody. Entire scFv gene-vector plasmid was transferred to CHO cell so as to produce scFv entire antibody. Cell culture medium ELISA test results showed negative. Condition optimation may be needed. In the study, we have set a basis for further study, including expression optimation, animal test and exploratory development.So far, some reports on screening and characterization of human scFv antibody against Aβpeptide have been found, but they are in phase of lab research. The report of successful clinical use of anti-Aβhumanized antibodies and entire antibody preparation hasn’t been found. NCBI blast shows our obtained anti-Aβhumanized antibody gene sequence is a new sequence. Our study will be a powerful tool for AD immunotherapy and clinical use study.