【作者】 杨涛；

【导师】 吴博威； 牛勃；

【作者基本信息】 山西医科大学 ， 生理学， 2004， 博士

【摘要】 抗肿瘤坏死因子α(TNF-α)抗体能够中和高浓度TNF-α在机体内产生的各种有害效应，阻止其引起的如类风湿性关节炎等自身免疫性疾病的发生和发展，在治疗多种相关疾病方面具有广阔的临床应用前景。已有许多实验证据表明，具有中和作用的抗TNF-α单克隆抗体能够预防和治疗因TNF-α过量而导致的炎症、感染性休克等相关疾病状态。我们利用噬菌体表面展示技术制备基因工程抗TNF-α单链抗体(scFv)，一方面建立了在体外大量制备抗体的简便的生产工艺，解决了目前TNF-α单抗制备工艺复杂、产量低的问题；另一方面由于scFv只含有抗体的重链可变区(VH )和轻链可变区(VL)片段，具有分子量小、免疫原性低、穿透力强的优点，克服了鼠源性单抗免疫原性强、分子量大、不易穿过血管壁的缺陷，使得抗TNF-α scFv在临床免疫治疗中具有良好的应用前景；第三，由于噬菌体展示技术实现了蛋白质表型和基因型的统一，使得该体系成为探索和分析蛋白质相互识别规律的一种极有价值的实验工具。本研究内容包括抗TNF-α噬菌体scFv抗体文库的构建，从中筛选、鉴定抗TNF-α scFv，用基因工程方法提高其表达量并进行人源化改造等工作，并对scFv重链可变区一级结构的保守性进行了初步分析。I 构建抗TNF-α噬菌体抗体scFv文库为了得到抗TNF-α scFv，我们首先构建了小鼠噬菌体单链抗体库。用rhTNF-α免疫小鼠后，间接ELISA方法测定免疫小鼠血清的抗体效价。结果表明其中两只小鼠的抗体效价介于10-5-10-6之间，符合构建抗体库的要求。分离免疫小鼠的脾细胞，提取总RNA，反转录成cDNA。以cDNA为模板，用重叠延伸PCR方法扩增得到scFv基因。此过程包括：①利用针对抗体重链和轻链可变区的引物分别扩增VH 和VL基因片段，将两种PCR产<WP=7>物进行纯化并测定浓度。②再用含Linker的引物将VH 和VL基因片段通过Linker连接起来形成scFv。③最后用含有相应酶切位点的引物扩增scFv，在scFv的5’端和3’端分别加上Sfi I和Not I酶切位点。经琼脂糖凝胶电泳分析，扩增的VH和VL基因片段的长度分别是360和330 bp左右，scFv基因片段的长度大约是750 bp。将scFv扩增片段纯化后，分别用Sfi I和Not I进行酶切，并与线性化噬粒pCANTAB 5 E体外连接。经限制酶酶切鉴定证实scFv片段以Sfi I/Not I位点定向插入噬粒的gpIII基因前方，将连接产物命名为pATF。用CaCl2法将连接产物转化感受态E.coli TG1，制备细菌形式的噬菌体单链抗体库，增加转化次数以提高抗体库的容量。通过菌落计数得到该抗体库的容量大约是4.6(106，大小中等。限制性酶切分析抗体库的重组率为83%。II 筛选抗TNF-α scFv阳性克隆及其鉴定我们利用rhTNF-α对抗体库进行筛选以得到TNF-α特异性噬菌体克隆。首先用rhTNF-α对抗体库进行3轮淘洗。用辅助噬菌体M13KO7感染转化菌，以挽救出噬菌体形式的抗体库。将此抗体库加入用rhTNF-α包被的酶标板内，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孔内，然后用pH 2.2的Tris将结合的噬菌体洗脱下来，感染对数生长期E.coli TG1，以扩增抗原特异性噬菌体克隆。重复上述操作两次。这样，经过三轮“吸附－洗脱－扩增”的淘洗过程，抗体库中与TNF-α特异性结合的噬菌体克隆从第一轮后的3.5(104 pfu增加至第三轮后的6.0(108 pfu，被富集了17 000倍。Dot blot检测也显示了明确的富集效果。随机挑取40个克隆，用M13KO7感染后使scFv表达于噬菌体表面，ELISA筛选呈现有抗TNF-α scFv的噬菌体克隆。检测结果表明，有3个克隆的A490值最高，对这3个克隆重复ELISA检测发现均具有特异性抗原结合活性，而且与其它抗原没有交叉反应。其中有1个克隆(B18)结合抗原的灵敏度最高，因此选择该克隆作进一步研究分析。III 抗TNF-α scFv的可溶性表达及鉴定在噬粒结构基因中，位于scFv和gpIII基因之间有一个琥珀终止密码<WP=8>子(TAG)，当噬粒在抑制性E.coli TG1中表达时，TAG被通读而不起终止密码子的作用，scFv与gpIII以融合形式表达并展现于噬菌体颗粒表面。而在非抑制性E.coli HB2151中，TAG被识别为终止密码子，scFv以可溶性方式分泌至细菌胞周质中。我们将筛选得到的抗TNF-α噬菌体阳性克隆感染E.coli HB2151，建立抗TNF-α scFv可溶性表达体系并对其进行活性检测。将阳性克隆感染E.coli HB2151，30(C、IPTG诱导表达20 h，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32(103，浓集于胞周质中，表达量占全菌总蛋白1%左右，将此可溶形式的抗TNF-α scFv命名为s-B18。用抗E-tag抗体亲和层析方法纯化位于胞周质中的s-B18，纯化后s-B18的回收率为0.3%，产量为3.3 mg/L。s-B18的活性测定采用以下方法：①竞争ELISA测定s-B18与TNF-α的亲和常数；②竞争ELISA检测s-B18对抗TNF-α IgG与TNF-α结合的抑制作用；③检测s-B18对TNF-α致L929细胞毒的中和作用；④Dot blot鉴定s-B18识别TNF-α表位的情况。结果表明，s-B18与TNF-α的亲和常数约为8(107 M-1，能够抑制抗TNF-α IgG与TNF-α的结合，且可以中和TNF-α对L929细胞产生的毒性作用，IC50为70 (g/ml。s-B18能够识别TNF更多还原

【Abstract】 Anti tumor necrosis factor α(TNF-α) antibody can neutralize the deleterious effects caused by high-level TNF-α in vivo. It can also block the pathway of autoimmune diseases such as rheumatoid arthritis (RA) etc. This provides it a broad clinical application in treating TNF-α related diseases. Many experiments have shown that anti-TNF-α monoclonal antibody (mAb) is effective in reducing the symptoms of inflammation and infectious diseases that are related with high-level TNF-α. Here we report the preparation of anti-TNF-α scFv using phage display technique. First, this establishes a massive and simply productive technology of antibody in vitro and resolves the problems of complicated production of anti-TNF-α mAb with low yield. Secondly, with respect to the shortcomings of antigenicity, large molecular mass and powerless penetration of murine mAb, scFv is characterized by its low mass, reduced immunogenicity and powerful penetration due to its structure composed of only VH and VL. This makes anti-TNF-α scFv a wide application in clinical immunological treatment. Thirdly, the physical association of the phenotype and genotype of phage display technique allows it a very valuable tool for exploitation and analysis of laws of protein recognition. In this paper, we present the construction of anti-TNF-α phage scFv antibody library and the isolation, characterization and humanizaiton of specific scFv directed against TNF-α. The expression level of anti-TNF-α scFv was improved using gene engineering technique and the consensus of primary structure of scFv VH was analyzed briefly. <WP=12>I Construction of anti-TNF-α phage antibody scFv libraryWe construct the mouse phage scFv library to obtain anti-TNF-α scFv. First, immunizing mice with rhTNF-α and the titer of antibody in sera was detected using ELISA. The results showed that the titer of antibody of two mice was up to 10-5-10-6 and conforms to the demands of phage antibody library construction. Then total RNA was isolated from splenocytes of these two mice and converted into cDNA. And scFv gene was amplified by the overlap extension PCR method using cDNA as template. This procedure included: ①The VH and VL fragments were separately amplified using the primer pairs that hybridize to the sequence encoding the variable regions of heavy and light chain. The PCR products were purified and the concentrations were determined. ②The VH and VL fragments were assembled into scFv with a specially constructed DNA linker by PCR. ③ScFv was amplified with 5’ heavy chain primer with Sfi I site and 3’ light chain primer with Not I site. After the analysis of agarose gel electrophoresis, the PCR product of VH formed a band at ~360 bp and that of VL at ~330 bp and the VH and VL were successfully spliced together to generate a fragment of scFv with 750 bp in length. The purified scFv was digested with Sfi I and Not I and cloned into phagemid pCANTAB 5 E. The restriction analysis of the ligation product suggested that scFv fragment was inserted in front of gIIIp of phagemid pCANTAB 5 E. Then the ligation product was named pATF and transferred into competent E.coli TG1 to generate a bacterial form of phage scFv antibody library. The transformation reaction was carried out several times to increase the diversity of the library. The diversity of the library evaluated by means of bacterial colony counts was about 4.6(106. It is medium. The recombinant ratio of the library was 83% by restriction analysis.II Screening and determination of anti-TNF-α positive phage clone The library was screened by rhTNF-α to get the antigen-specific phage clones. Above all, three panning rounds were performed of the library with rhTNF-α. The transformed cells were infected with M13KO7 helper phage to produce a <WP=13>phage form of library. Then the library was added into microtiter wells coating with rhTNF-α and incubated for a period of time and washed several times. The phage clones binding specifically to TNF-α were retained in the wells. These clones were eluted from the we更多还原