【作者】 包福祥；

【导师】 曹贵方； 何金生；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2009， 博士

【摘要】 老年性痴呆(Alzheimer’s disease,AD)是一种渐进性神经退行性疾病,其病理特征是渐进性的老年斑沉积,神经突触丢失和神经纤维缠结,临床表现为记忆功能衰减及识别能力障碍,同时伴有各种神经症状和行为障碍。在老年人群,具有非常高的发病率,目前还没有特异性治疗药物。近年来AD的发病机理和药物研究方面都有突破性进展,尤其是制备针对β淀粉样蛋白(amyloid beta protein,Aβ)特异性抗体药物成为AD治疗极具价值的途径。噬菌体展示技术(phage display)为抗体制备开创了一条简便、快速的基因工程抗体制备方法。噬菌体抗体是在噬菌体表面表达抗体分子的抗原结合片段(Antigen binding fragment,Fab)或单链抗体(single chain fragment variable, scFv),单链抗体具有分子量小,免疫原性低,穿透力强,定位迅速,易于基因工程制备和改造等优点。通过构建和筛选人源性天然噬菌体抗体库,得到Aβ寡聚体特异性人源性单链抗体,可直接应用于人AD的治疗。利用噬菌体展示技术构建人源性天然抗体库,以可溶性Aβ1-42寡聚体对抗体库进行筛选获得针对低分子量Aβ1-42寡聚体的特异性单链抗体。分离10个健康人外周血淋巴细胞,提取总RNA,RT-PCR法得到cDNA链,以cDNA链为模板扩增得到全套人抗体VH和VL基因,经过重叠延伸PCR将VH和VL连接得到scFv片段,将scFv片段酶切后克隆至pCANTAB5E噬菌体载体,电转化TG1感受态菌,经辅助噬菌体M13K07拯救,获得库容为2.5×109单链抗体库。以可溶性Aβ1-42寡聚体为抗原,对抗体库进行四轮筛选,ELISA法筛选特异性识别Aβ1-42寡聚体的阳性克隆,将筛选到的阳性克隆B19转化至E.coli HB2151,诱导表达可溶性scFv。SDS-PAGE及Western Blot分析结果显示可溶性scFv获得了正确表达,且能够与Aβ1-42三聚体及纤维特异性结合,可溶性scFv与Aβ1-42寡聚体的亲和常数为9×10-6 mol/L。将单链抗体的重链和轻链可变区基因克隆入原核表达载体pComb3C/cFab中,转化TOP10感受态,诱导表达了重组抗体B19-Fab。SDS-PAGE及Western Blot分析结果显示,重组抗体B19-Fab得到了正确的表达。ELISA结果显示,重组抗体B19-Fab仍然保持对Aβ1-42寡聚体特异性结合活性。对单链抗体亲和力成熟进行的初步研究为后续的打下了良好的基础。本实验中获得的Aβ1-42寡聚体特异性单链抗体为老年性痴呆症(AD)的临床治疗研究奠定了基础。更多还原

【Abstract】 Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the elder. The disease is characterized by memory impairment and cognitive obstacle. The current treatments of AD provide only symptomatic relief and are practically inefficient. Now, anti-Aβantibodies have been suggested as a potential therapeutic strategy for the treatment of AD.Genetic engineering made it possible to generate antibody without constant region which is termed scFv(single chain fragment variable) or Fab (Antigen binding fragment). ScFv is a smaller functional unit remaining antigenic specificity and affinity. Compared with other antibodies, scFv has many advantages, smaller size, reduced immunogenicity, stronger penetrating power, rapid localization and being easily cleared up from the normal tissue. Meanwhile, scFv can be produced in large scale in bacteria or yeast and manipulated by genetic engineering. Therefore, developing scFv is of great significance in the areas of AD diagnosis and therapy.To construct the human single-chain Fv (scFv) antibody library by phage display technology and obtain the specific scFvs (single-chain fragment variable) against soluble Aβ1-42 (Amyloid-beta ) oligomers by screening the human scFv library, peripheral blood lymphocytes (PBL)were separated from ten healthy donors, and the total RNA was extracted from the PBL. The variable heavy(VH) and variable light(VL) genes were amplified by RT-PCR and then the scFv was obtained through SOE-PCR. The scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent E.coli TG1 cells, and then a scFv phage display library containing 2.5×109clones was gained. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Aβ1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E.coli HB2151 to express soluble scFv. The soluble scFv antibodies were expressed successfully, and displayed specific binding to Aβ1-42 trimer and protofiber by SDS-PAGE and Western blot analysis.The binding affinity of soluble scFv antibodies to Aβ1-42 oligomers was 9×10-6 mol/L. For the purpose of affinity maturation, the VH and VL gene fragments were cloned into the pComb3C/cFab vector, and transformed to competent E.coli TOP10. SDS-PAGE and Western blot analysis confirmed that the soluble B19-Fab fragments were expressed successfully, and preserving the binding affinity to Aβ1-42 oligomers. The preparation of specific scFv against Aβ1-42 oligomers can be used further in the therapeutic research on AD.更多还原