【作者】 夏汝山；

【导师】 刘玉峰；

【作者基本信息】 第四军医大学 ， 皮肤病与性病学， 2004， 博士

【摘要】 研究表明，人体内存在的天然抗角蛋白自身抗体(anti-keratin auto antibody，AK auto Abs)存在于人和多种动物体内，构成了免疫系统的基本组成部分，参与免疫自稳态的调节。在一些病理情况下(如银屑病、接触性皮炎等)，AK auto Abs的存在有利于限制病情进展，促进损伤修复，对机体具有保护作用。本室发现AK auto Abs主动介入某些皮肤病的病理生理过程，无论是来自皮损或者正常人血清的AK auto Abs都以剂量依赖的方式抑制角质形成细胞的增殖和分化；输注含高滴度AK auto Abs的人血丙种球蛋白，在部分银屑病患者收到了较好的效果，从血清中提取的AK auto Abs在动物实验中也初步显示了有希望的治疗应用前景，但由于血液制品的来源和安全性问题而限制了AK auto Abs的临床应用。 噬菌体展示技术的出现为不经免疫制备人源性抗角蛋白抗体提供了可能。国内外已经从噬菌体抗体库中成功地制备了多种单克隆抗体，本实验室从半合成的噬菌体抗体库中成功地筛选到抗角蛋白的噬菌体抗体Fab和ScFv，并初步得到可溶性表达的抗角蛋白单克隆抗体Fab，其具有较高的特异性和亲和力，为抗角蛋白NAA的应用及进一步研究提供了良好的手段。研第四军医大学博士学位论文究发现，这些抗体片段具有抑制培养的角质形成细胞增殖的作用，从噬菌体抗体库中筛选出来的抗体分子在很多情况下可满足应用的需要，但由于没有Fc段而不具备抗体的某些生物学功能，如激活补体、ADCC(抗体依赖性细胞介导的细胞毒作用)以及免疫调节等。在某些情况下，尤其用于体内治疗时，需要使用完整的抗体分子。亲和力是抗体的重要参数，在体内只有亲和力高的抗体才能发挥有效的生物学作用。在生物技术领域，亲和力高的抗体有着明显较高的使用价值，因此，对于从噬菌体抗体库筛选出的抗体，只有及时评价抗体的亲和力，才能选择出性能优良的抗体。为此我们选择亲和力最高的Fab或scFv克隆，克隆抗体的可变区基因，构建真核表达载体，脂质体Lipofeetamine2000转染二氢叶酸还原酶(DHFR)缺陷的CHO细胞，在MTx加压条件下，表达抗人角蛋白全长IgG抗体，并检测抗体的生物学活性。 本室己经从半合成噬菌体抗体库筛选出7株抗角蛋白ScFv和4株抗角蛋白Fab。通过ELISA检测抗体活性及特异性，并进行了研怂stem blot分析。筛选亲和力最高的抗体克隆尤其重要，而目前通常采用硫氰酸胺法或生物传感器来测定抗体的相对亲和力，一由于噬菌体ScFv切除基因m后不能自连，因此不能表达出抗体片段，也无法预知抗体的活性，不能应用生物传感器测定抗体的亲和力。因此我们利用曾经筛选到的4株抗角蛋白噬菌体Fab抗体片段，通过内切酶NFhel切除载体上的基因111，IPTG诱导表达可溶性抗体Fab片段，应用镍亲和层析柱纯化抗体，生物传感器测定抗体亲和力，选择亲和力最高的克隆。亲和力最高的克隆质粒送上海生物工程公司测序，根据测序结果和前导序列设计扩增抗体轻、重链可变区(VK和VH)基因以及连接前导序列的引物。经过PCR扩增带有前导序列的轻、重链可变区基因(LK和LH)。经Xbal和BamHI酶切LK，分别插入同样酶切的载体pWD和pWSD3，构建带有LK的载体pWD、和pWSD3K;经Xhol和Hind 111酶切LH，分别插入同样酶切的载体pWDK和pWSD3K，构建带有目的基因轻、重链抗体可变区基因的真核表达载体pWD出和pwSD3妞。分别进行酶切、PcR和测序鉴定正确:经LipofectamineZo00转染CHo(dhfr一)细胞，72小时后检测抗体的瞬时表达;在氨甲喋吟伽TX)加压筛选条件下，亚克隆化筛选稳定表达的细胞第四军医大学博士学位论文系，Rl’- pCR检测抗体在mRNA水平的表达，ELISA检测抗人角蛋白全长IgG抗体的表达，W七stem blot分析抗体识别角蛋白的相对分子质量;收集表达的上清液，作用于培养的鳞癌细胞，MTT检测表达的全长IgG抗体对细胞增殖的影响。 本实验发现，7株人抗角蛋白噬菌体SeFVHAKSS、HAKS 19、HAKS 21、HAKS 24、HAKS 28、HAKS 31和HAKS 41能特异性识别人角蛋白，而与转铁蛋白、胰蛋白酶、人工gG、TRP一1等抗原无交叉结合反应，其识别角蛋白的相对分子质量约在56 000一57 000之间，包括角蛋白K5和K6;DNA测序表明HAKSS、19和21的VL属人19入链第二亚群，HAKS 24和28的VL属人Ig入链第一亚群，HAKS31和41的vL属人IgK链第三亚群;HAKSS的重链可变区基因(vH)属人IgG第一亚群，噬菌体HAKS 19、21、24、24、31和41的VH属第三亚群。其中HAKS 31和41的轻重链完全一样，是同一个克隆;经过硫氰酸胺检测，HAKss和HAKS19的相对亲和力最小，较接近;HAKS28、31和HAKS41的相对亲和力最大，比较一致;HAKS 21和HAK24的相对亲和力较接近;7株角蛋白噬菌体scFv的相对亲和力由大到小为:HAKS 31二HAKS 41二HAKS 28>HAKS艺1、HAKS 24)HAKSS、HAKS 19;由于噬菌体ScFv不能表达出可溶性抗体片段，无法应用生物传感器较精确地测定抗体的亲和力。因此我们利用曾经筛选到的4株抗角蛋白噬菌体Fab抗体，通过表达和纯化可溶性抗体Fab片段，应用生物传感器测定抗体的相对亲和力，在4株抗角蛋白噬菌体Fab片段中来选择亲和力最高的抗体，结更多还原

【Abstract】 As some investigations have shown that human anti-keratin auto antibody (AK auto Abs) generally exist in human and animal bodies, and compose of the basic part of immune system. AK auto Abs play an important role in immune homeostasis. In some pathological states such as psoriasis and contact dermatitis, the existence of AK auto Abs could inhibit the progression of the diseases and be beneficial for the recovery from the diseases. Animal experiments have proved AK auto Abs are involved in the pathophysiological process of some dermatogic diseases. The purified AK auto Abs inhibited the proliferation and differentiation of keratinocytes depending on the dosage. The infusion of human serum immuglobin containing AK auto Abs had obvious results in some psoriasis patients. The AK auto Abs which were extracted from animal serums showed the prospective therapy and prospective application. But The clinical application of AK auto Abs was limited by the reliability of blood agents.The phage-display technology makes us to be possible to produce human antikeratin antibodies not by means of immune methods. Many monoclonal antibodies were madesuccessfully from phage antibody libraries in home and abroad. Recently our laboratory has already screened out the Fab and ScFv of anti-keratin antibodies successfully from semisynthetic phage antibody library and obtained soluble Fab with high specificity and affinity which provides better means for the application and further study of AK auto Abs. In our research Some fragments of these antibodies have the function of inhibiting proliferation of cultured keratinocytes. The antibody fragments, which were screened out from phage antibody libraries, can meet the needs of application. But some biological functions, such as complement activation, antibody-dependent cellular cytotoxicity (ADCC) and immunoloregulation, are lost because of not having Fc fragments. In some conditions especially treatment in vivo, the full length of IgG antibody molecule is needed. So we select the Fab or ScFv with the highest affinity to clone the variable region genes and construct the eukaryotic expressive vector which is transfected to dihydrofolate reductase defective CHO cells with lipofectamine 2000. Under the pressure of MTX, the transfectants express the human anti-keratin full-length of IgG antibody and its biological activity is detected.Affinity is an important parameter of antibody. It is that the antibody with high affinity can play an effective biological role in vivo. In biological and technological field, the antibodies with high affinity have advantageous value in clinical application. So in order to select the antibody with good behavior, we should elevate the affinity of antibodies which is screened out from phage display library timely. Our laboratory have screened out seven anti-keratin single chain antibodies and four anti-keratin Fab antibodies. Their activity and specificity is detected with enzyme-linked immunoadsordent assay. The relative molecular weight of antigen binding is analysed with western blot. But it is the most important to estimate the affinity of antibody by sulforhodanide or biosensor and screen the antibody clone with the maximinal affinity. Phage single chain antibodies can not bond themselves. So we cannot express the antibodies fragment, predict the activity of antibodies and detect the affinity of antibodies with biosensor. So we utilized the four strains of anti-keratin phage Fab antibodies to induce the expression of soluble antibodies with IPTG. The antibodies were purified with the affinity with the nickel column of affinity chromatography and the affinityis measured with biosensor. We selected the antibody with the maximal affinity.The plasmid with the maximal affinity is sequenced in Shanghai biological engineering Corporation. We design the primers according to the sequencing results to amplify the variable region genes of light and heavy chain and connect the leader sequence. The variable region genes of light and heavy chain with leader sequences(更多还原