【作者】 吴炜霖；

【导师】 仲人前； 孔宪涛； 韩焕兴； 邓安梅；

【作者基本信息】 第二军医大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 研究目的:自身免疫性疾病(autoimmune diseases, AID)是指机体对自身抗原发生免疫反应而导致自身组织损伤所引起的疾病,是一类较常见的难治病。其突出特点是患者体内产生高滴度自身抗体,并通过免疫复合物等途径,产生针对自身组织的免疫病理性损伤,累及一个或全身多个器官、系统。自身免疫病发病机制十分复杂,涉及因素众多。一般认为以免疫活性细胞数量和功能的失常,导致免疫功能紊乱,从而产生大量自身抗体为主要机制,但具体机制并不明确。因而自身抗体的研究往往成为对自身免疫性疾病研究的关键点和切入点。目前,自身免疫病的诊断手段非常有限,主要是在结合临床的基础上,依赖实验室检查,在AID患者体内检测到一种或多种高效价的自身抗体为诊断该病的重要依据。自身免疫病缺乏特异的早期诊断指标及特效的治疗药物,这都直接影响了疾病的诊断和治疗。因此,获得纯化的可以满足不同需要的高亲和力、高特异性自身抗体对于研究自身免疫性疾病的发病机制及临床诊疗都具有非常重要的价值。抗体的制备工艺历经100多年的发展,经历了从多克隆抗体、单克隆抗体到人源化抗体的不同阶段。在人源单克隆抗体的制备技术中,噬菌体抗体库技术是迄今发展最成熟、应用最广泛的抗体制备技术之一。利用此技术,外源蛋白或多肽的基因与噬菌体外壳蛋白的基因融合,外源产物表达在噬菌体的表面,从而实现了基因型与表现型的完美统一。目前,国内外已成功构建了针对乙肝病毒、肿瘤细胞等多种抗原的噬菌体抗体库,并从中筛选得到具有较高亲和力和特异性的抗体,潜藏着巨大的应用价值。本课题采用噬菌体抗体库技术,以50多例自身免疫病患者的外周血淋巴细胞为原料,构建出自身免疫性疾病的噬菌体抗体库,并以确定的自身抗原dsDNA作为固相抗原,从抗体库中筛选、富集出相关抗体,以鉴定所构建抗体库的有效性,为进一步利用该抗体库筛选出不同需要的抗体及其功能研究提供平台。研究方法:1.从55名自身免疫性疾病患者(包括SLE、RA、强直性脊柱炎、干燥综合征、PBC等)外周血中分离出单个核细胞,抽提总RNA,反转录生成cDNA,以内参GADPH验证混合cDNA的质量;2.以上述cDNA为模板PCR扩增轻链和重链基因;3.挑取XL1-Blue的单克隆菌落,采用Hepes法制备感受态细胞;4.将轻链基因克隆入pComb3Hss载体以构建轻链库;5.将重链基因载入重组轻链库,构建组合文库H+L+pComb3Hss;6.将组合文库转化大肠杆菌XL1-Blue,加入辅助噬菌体M13KO7,随机的组合文库表达于噬菌体表面,完成噬菌体表面展示;7.用商品化anti-dsDNA ELISA试剂盒,对噬菌体抗体进行4轮亲和富集,测定抗体库滴度;8.间接ELISA法鉴定4轮淘选后的噬菌体抗体,提取阳性克隆的的噬菌粒,切除gIII片段,自连接后转化XL1-Blue,以IPTG诱导可溶性Fab片段的表达;9.分别用anti-dsDNA和anti-U1RNP检测试剂盒对制备好的Fab片段进行ELISA检测,鉴定其抗原特异性。结果:1.抽取总RNA 7-10ug,抽提的RNA质量可靠,经GADPH验证的cDNA质量可靠;2. PCR扩增了大小为660bp的轻链κ、λ基因及重链Fd基因,并成功构建了重组轻链库,菌落计数得轻链库的库容约4×105,酶切鉴定重组率为80%;3.同法测得噬菌体抗体库的库容为6×106,酶切鉴定重组率为70%,PCR鉴定证明轻重链均已转入,噬菌体抗体库构建成功;4.用抗原固相法对噬菌体抗体库进行了4轮亲和富集,第1轮洗脱的滴度为1.1×105,第2、3轮都较前有较大提高,第4轮产出率比第1轮加了218倍;5.通过固相化抗原吸附法筛选获得2个阳性克隆,切除gIII基因,实现了可溶性Fab片段的表达;6.间接ELISA法检测显示6株Fab片段克隆均呈dsDNA阳性,U1RNP阴性,证实获得的可溶性抗体Fab片段具有特异性抗原结合活性。结论:利用自身免疫性疾病患者的外周血单个核细胞制备了混合cDNA,并在此基础上成功构建了自身免疫性疾病的噬菌体抗体库,经亲和富集后实现了抗体Fab段的可溶性表达,并具有较强的抗原特异性,因此该抗体库的建立为进一步筛选出其他高亲和力的自身抗体及其功能研究打下了基础,提供了良好的库源。更多还原

【Abstract】 Autoimmune disease means body’s immune response against autoantigen which led to self-tissue injury, it is common but difficult to treat. Its salient features are multiple autoantibodies, and immune pathological damage against self-tissue by ways such as immune complexes, resulting in injury almost all over the body. The pathogenesis of autoimmune diseases are complex, involving many factors such as genetic, infection, sex hormones, immune dysfunction. So far, there are many questions about autoimmunity don’t have answers, for example, how to start autoimmunity, the genetic background , the damage mechanism and immunologic recognition of autoimmunity, and the pathopoiesis of autoantibodies. Generally speaking, autoimmune disease is caused by the number abnormality and dysfunction of immune cell, which led to immune functional disorder, followed by the appearance of multiple autoantibodies, but the specific mechanism of autoimmune disease is still unknown. The research on autoan tibodies is always the key point of AID.Now, the diagnosis of autoimmune disease is limited, the main diagnostic code is the clinical symptom together with one or more high titer autoantibodies. The acknowledged specific antibodies of AID are seldom, while some adjunct antibodies are not specific for AID. Further more, the detection level of AID in China is varied, and there isn’t a specific index for early diagnosis, as well as specific drug, all of these have influence on diagnosis and treatment of AID. Therefore, purified antibodies with high affinity and high specificity have great value in our research on autoimmune disease.The preparation and purification of antibody have become the focus of immunology for many years, and the development of antibody have been under way for more than 100 years, which were divided three periods , namely, the period of polyclonal antibody, the period of monoclonal antibody and the period of humanized antibody. So far, the phage antibody library technology is the most sophisticated, the most widely used technology for antibody humanized. With the help of this technology, the gene of foreign protein or polypeptide was fused with the gene of coat protein of phage, so that the foreign protein or polypeptide expressed on the surface of the phage, achieving the unity of genotype and phenotype. Until now, several phage antibody library against HBV, tumor cells and so on have been successfully constructed, and antibodies with high affinity and high specificity were obtained. These antibodies were of great value.In this study, we used peripheral blood mononuclear cell of 55 autoimmune disease patients to construct a phage antibody library against AID, and obtained antibody against double-stranded DNA by panning and solid-phase antigen assay. Our study hope to lay the foundation for further panning and function research.Methods:1. The peripheral blood mononuclear cell of 55 autoimmune disease patients were isolated, then total RNA was extracted and reverse transcription to cDNA .The quality of cDNA mixture was verified by reference gene GADPH;2. By using the template cDNA verified above, human immunoglobulinκ/λlight chains and heavy chains Fd genes were amplified by polymerase chain reaction (PCR);3. The single colone of XL1-Blue was picked to prepare the competent cell by using Hepes solution;4. The light chain genes were first cloned into pComb3Hss vector to construct a human recombinant light chain library;5. The heavy chain genes were subsequently inserted into the corresponding sites of recombinant light chain library to generate a combinatorial H+L+pComb3Hss plasmid;6. The combinatorial plasmid transformed into E.coli.XL1-Blue, then E.coli.XL1-Blue was infected by helper phage M13KO7. A random combinatorial library was expressed on the surface of filamentous phage;7. The phage antibody library were panninged for four rounds by using anti-dsDNA ELISA kit, determinated the titer after each round;8. Identified the phage antibodies after four rounds panning by indirect ELISA. Plasmid DNA isolated from positive clone was cut off gIII genes. After ligated itself, the recombinant plasmid transformed E.coli.XL1-Blue, then XL1-Blue was induced by IPTG to product soluble Fab.9. Finally, the antigenic specificity of soluble antibody Fab was identified by anti-dsDNA ELISA kit and anti-U1RNP ELISA kit.Results:1. The total RNA extracted was 7-10 ug, and the quality of RNA was good. The cDNA was verified well, too;2. Human immunoglobulin light chainsκ,λand heavy chain Fd genes (MW 660bp) were obtained by PCR successfully. The constructed light chains library size is 4×105 and the recombination frequency is 80 percent;3. The constructed heavy chains library size is 6×106 and the recombination frequency is 70 percent, PCR showed that both light chain and heavy chain are inserted in the recombinatorial library and the library was successfully constructed.4. The titer of eluted phage after the first bound was 1.1×105 , and the titer after both the second and the third bound raised remarkably, the eluted phages were enriched 218 fold after the forth rounds panning.5. Two unique clones were isolated from the human Fab fragment library. It was proved the recombinant plasmid had been cut off gIII genes and ligated by itself through electrophoresis after digested by XhoI.6. The results of indirect ELISA showed that all of six clones of Fab were dsDNA positive while U1RNP negative, indicating the soluble antibody Fab had specific to bind with dsDNA.Conclution:We gained cDNA mixture successfully by reverse transcription technology from total RNA of peripheral blood mononuclear cells of fifty-five autoimmune disease patients with high titer autoantibodies and antinuclear antibodies in the serum. A human antibody Fab fragment library against AID was constructed based on it. Subsequently, human Fab fragment phage antibody library against double-stranded DNA was constructed successfully by phage display technology. Through four rounds panning of the antibody library, soluble Fab fragment was obtained and identified antibody activity against double-stranded DNA. The construction of phage antibody library against AID lay the foundation for the further panning of more autoantibodies with high affinity as well as their function research.更多还原