人源性TRAb Fab段抗体库的构建筛选与鉴定

发布时间：2018-06-04 10:33

  本文选题：噬菌体表面展示 + 人源性单克隆抗体　； 参考：《天津医科大学》2012年博士论文

【摘要】：背景和目的Graves病患者血清中的促甲状腺素受体抗体(TRAb)分为:(1)TSH受体刺激性抗体(TSAb);(2)TSH受体刺激阻断性抗体(TSBAb);(3)中性TSH受体抗体。随着基因工程技术的发展,运用噬菌体展示技术,通过对人源性促甲状腺素抗体Fab片段抗体库的构建筛选、鉴定,得到TRAb单克隆抗体,为进一步建立TRAb的定量分析方法及探索免疫干预治疗奠定基础。方法(1)从血清TRAb浓度大于40IU/L的自身免疫性甲状腺疾病患者外周血中,抽提总RNA,反转录获取cDNA。以获取的cDNA为模版,PCR法扩增免疫球蛋白分子轻链λ、λ基因及重链Fd基因。将轻链克隆入pComb3Hss载体以构建轻链文库。将重链基因载入κ/λ-pComb3Hss载体,构建完成组合文库。(2)将重组质粒转化大肠杆菌XL1-Blue,用辅助噬菌体M13K07感染,随机的组合文库表达于丝状噬菌体表面,完成噬菌体表面展示。(3)应用固相化抗原(TSHR-N)吸附筛选法通过数轮"吸附—洗脱—扩增"富集噬菌体抗体,筛选阳性克隆。(4)取阳性克隆的噬菌体DNA,切除gⅢ基因片段,自连接后转化大肠杆菌XL1-B1ue,以IPTG诱导表达可溶性TRAb Fab片段,应用间接ELISA法对表达产物进行鉴定。(5)利用亲和层析柱纯化人源性TRAb Fab片段,Western blotting鉴定纯化产物。(6)对表达较好的阳性克隆进行测序分析,轻链和重链可变区的DNA序列采用双向测序。结果(1)从外周血单个核细胞中抽提得到总RNA,并成功反转录获得cDNA文库。(2)PCR法扩增了大小均为680bp左右的轻链κ、λ基因及重链Fd基因,并成功构建了库容为1.32 × 105基因抗体库(轻链库)和库容为2.28 × 1 05Fab抗体库(组合文库)。(3)通过噬菌体表面展示技术,在辅助噬菌体M13K07的帮助下,在组合文库的基础上获得了富含TRAb Fab片段的人源性噬菌体抗体库。(4)以TSHR-N为抗原,通过固相化抗原吸附筛选法对构建的Fab噬菌体抗体库进行5轮富集筛选,初步成功筛选到特异性TRAb Fab噬菌体抗体,富集效应约77倍。(5)通过Phage-ELISA检测,结果鉴定出了具有抗原结合活性的单克隆。提取阳性克隆的噬菌体DNA,切除gⅢ基因片段,实现了可溶性人源性TRAb Fab片段的表达。(6)利用Ni2+金属敖合层析法获得纯化的人源性TRAb Fab片段。(7)间接ELISA法检测结果显示:制备的可溶性人源性TRAb Fab片段具有特异性结合TSHR-N端的免疫学活性。(8)经GenBank检索DNA序列分析,证实该克隆的轻链可变区与人的免疫球蛋白λ链同源性达到94.4%,重链可变区与人的免疫球蛋白IgG重链VH4同源性为88.9%。结论本研究通过噬菌体展示技术成功构建了人源性TRAb Fab片段组合文库,并通过固相化抗原吸附筛选法筛选获得阳性克隆,实现了可溶性TRAb Fab片段的表达,基因测序证实其轻重链与人免疫球蛋白可变区具有同源性,ELISA结果显示其具有特异性结合TSHR-N端的免疫学活性。
[Abstract]:Background and objective Thyrotropin receptor (TSH) receptor antibody in the serum of patients with Graves's disease is divided into two groups: TSAB receptor stimulative antibody (TSAB) and TSH receptor stimulating blocking antibody (TSB). With the development of genetic engineering technology, TRAb monoclonal antibody was obtained by using phage display technology and the construction and identification of human thyrotropin antibody Fab fragment antibody library. To establish the quantitative analysis method of TRAb and to explore the immunological intervention therapy. Methods 1) Total RNAs were extracted from peripheral blood of patients with autoimmune thyroid disease whose serum TRAb concentration was higher than 40IU/L, and cDNA was obtained by reverse transcription. The immunoglobulin molecular light chain 位, 位 gene and heavy chain FD gene were amplified by using the obtained cDNA as template. The light chain was cloned into pComb3Hss vector to construct the light chain library. The heavy chain gene was loaded into 魏 / 位 -pComb3Hss vector, and the recombinant plasmid was transformed into Escherichia coli XL1-Blue. the recombinant plasmid was randomly expressed on the surface of filamentous phage by phage M13K07 infection. The phage surface display was completed. (3) the phage DNA of positive clone was screened by solid phase antigen (TSHR-N) adsorption screening method, and the g 鈪，

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