Aβ寡聚体特异性单链抗体的构建、表达和生物学功能研究

发布时间：2019-03-28 06:33

【摘要】：目的：阿尔茨海默病(Alzheimer's disease, AD)是一种引起老年人痴呆的最常见的神经退行性疾病,目前尚无有效的治疗方法。β淀粉样肽(P-amyloid peptide,Aβ)寡聚体是AD发生的早期始动因子。本研究拟在Ap寡聚体特异性单抗A8基础上,构建单链抗体(single chain fragmen variant, scFv),表达并初步探讨其生物学功能方法：以A8单抗轻重链可变区基因为模板,通过重叠延伸PCR的方法构建scFv的基因片段,同时引入不同的连接肽(Linker)序列,(G4S)3或者p2A,拼接得到多种形式的scFv片段。通过原核表达体系快速验证所构建单链抗体基因片段表达产物的活性,并且构建真核表达载体。脂质体法分别转染人宫颈癌细胞(Hela)和中国仓鼠卵巢细胞(CHO),潮霉素加压筛选,筛选稳定表达抗Ap寡聚体的单链抗体细胞株。通过间接ELISA和斑点印迹分析所得抗Aβ寡聚体scFv的抗原识别能力,通过体外实验研究所得scFv的细胞保护作用以及超微病理改变。结果：成功获得了scFv的3个基因片段,VL-(G4S)3-VH、VH-(G4S)3-VL和VL-p2A-VH。选取前2个基因片段构建原核表达载体,获得30kD的目的蛋白,经过镍亲和层析纯化,Western blot结果显示纯化的scFv正确表达；间接ELISA结果表明VH-(G4S)3-VL滴度为1：32000,VL-(G4S)3-VH滴度为1：8000。成功构建3个真核表达载体(pSecTag2/HygroA-VL-(G4S)3-VH、pSecTag2/HygroA-VH-(G4S)3-VL和pSecTag2/HygroA-VL-p2A-VH);获得了2株稳定表达scFv的细胞株Hela-VL-p2A-VH(?)(?)CHO-VL-(G4S)3-VH。同接ELISA和斑点印迹结果表明所分泌的细胞上清具有抗原识别能力,体外实验显示其具有阻断和抑制Aβ42寡聚体的细胞毒性作用。其中一细胞株Hela-VL-p2A-VH较CHO-VL-(G4S)3-VH表达量和识别能力相对强。结论：成功构建了Aβ寡聚体单链抗体表达载体,通过原核和哺乳动物细胞系统正确表达；建立了2株稳定表达所述单链抗体的细胞株；所分泌的细胞上清具有抗原识别能力,能够阻断Ap寡聚体的细胞毒性作用,为进一步应用奠定基础。
[Abstract]:Objective: Alzheimer's disease (Alzheimer's disease, AD) is one of the most common neurodegenerative diseases that cause dementia in the elderly. There is no effective treatment for Alzheimer's disease. Beta amyloid peptide (P-amyloid peptide,) A 尾) oligomer is the early initiation factor of AD. In this study, based on Ap oligomer specific monoclonal antibody A8, the expression of single chain antibody (single chain fragmen variant, scFv), was constructed and its biological function was preliminarily studied: the variable region of light and heavy chain of monoclonal antibody A8 was used as template. The gene fragment of scFv was constructed by overlapping extension of PCR. At the same time, different ligated peptide (Linker) sequences, (G4S) 3 or p2A were introduced, and various forms of scFv fragments were obtained by splicing together. The activity of the expressed product of scFv gene fragment was verified by prokaryotic expression system, and the eukaryotic expression vector was constructed. Human cervical cancer cell line (Hela) and Chinese hamster ovarian cell line (CHO), were transfected with lipofectamine to screen the single chain antibody cell line stably expressing anti-Ap oligomer. The antigen recognition ability of anti-A 尾 oligomer scFv was analyzed by indirect ELISA and dot blot analysis. The cell protection and ultrastructural changes of scFv were studied by in vitro experiments. Results: three scFv gene fragments, VL- (G4S) 3VH, VH-(G4S) 3-VL and VL-p2A-VH., were successfully obtained. The first two gene fragments were selected to construct the prokaryotic expression vector, and the target protein of 30kD was obtained. The purification of, Western blot by nickel affinity chromatography showed that the purified scFv was expressed correctly. The indirect ELISA results showed that the titers of VH- (G4S) 3-VL and VL- (G4S) 3-VH were 1? 32000 and 1? 8000, respectively. Three eukaryotic expression vectors (pSecTag2/HygroA-VL- (G4S) 3VH, pSecTag2 / Hygro VH-(G4S) 3-VL and pSecTag2/HygroA-VL-p2A-VH) were constructed successfully. Two Hela-VL-p2A-VH (?) CHO-VL- (G4S) 3 VH. stably expressing scFv were obtained. The results of co-linking ELISA and dot blot showed that the secreted supernatant had the ability of antigen recognition. In vitro experiments showed that it could block and inhibit the cytotoxicity of A 尾 42 oligomer. The expression and recognition ability of one cell line Hela-VL-p2A-VH was stronger than that of CHO-VL- (G4S) 3-VH. Conclusion: the A 尾 oligomer single chain antibody expression vector was successfully constructed and expressed correctly by prokaryotic and mammalian cell systems, and two cell lines stably expressing the single chain antibody were established. The secreted supernatant has the ability of antigen recognition, which can block the cytotoxicity of Ap oligomers and lay a foundation for further application.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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