稳定表达Aβ特异性单链抗体的哺乳动物细胞株构建和功能研究
发布时间：2018-10-31 17:21
【摘要】：构建可以稳定表达Aβ特异性单链抗体(scFv)的哺乳动物细胞株。应用重叠延伸PCR的方法,以前期建立的Aβ特异性单克隆抗体(A8)的轻、重链可变区基因为模板,构建scFv的基因片段,通过(G_4S)_3或p2A两种不同的连接肽(Linker)序列,拼接得到多种形式的scFv基因片段,用于构建真核表达载体。利用脂质体分别转染人宫颈癌细胞(Hela)和中国仓鼠卵巢细胞(CHO),Western blot鉴定scFv的表达情况;以潮霉素筛选获得稳定表达抗Aβ的scFv细胞株,以间接ELISA和斑点印迹分析所得scFv的抗原识别能力;采用体外细胞实验,在超微病理水平分析所得scFv的细胞保护作用。结果:成功构建了Aβ特异性scFv的3个真核表达载体pSecTag2/HygroA-VL-(G_4S)_3-VH、pSecTag2/HygroA-VH-(G_4S)_3-VL和pSecTag2/HygroA-VL-p2A-VH,获得了2株稳定表达Aβ特异性scFv的细胞株Hela-VL-p2A-VH和CHO-VL-(G_4S)_3-VH。Western blot结果表明了相应scFv的正确表达,间接ELISA和斑点印迹结果表明所分泌的细胞上清具有Aβ抗原识别能力,体外实验显示其具有阻断和抑制Aβ寡聚体细胞毒性的作用。稳定表达Aβ特异性单链抗体的细胞株有助于AD免疫治疗基础研究的进一步开展。
[Abstract]:Mammalian cell lines expressing A 尾 -specific scFv (scFv) were constructed. Using the method of overlapping extension PCR, using the light and heavy chain variable region of A 尾 -specific monoclonal antibody (A8) as template, the gene fragment of scFv was constructed by (GSP) _ 3 or p2A (Linker) sequence. A variety of scFv gene fragments were obtained by splicing to construct eukaryotic expression vector. The expression of scFv in human cervical cancer cell line (Hela) and Chinese hamster ovary cell line (CHO), Western blot was identified by liposome transfection. ScFv cell lines with stable expression of A 尾 were obtained by hygromycin screening, the antigen recognition ability of scFv was analyzed by indirect ELISA and dot blot analysis, and the cytoprotective effect of scFv was analyzed by cell experiment in vitro. Results: three eukaryotic expression vectors of A 尾 -specific scFv were successfully constructed: pSecTag2/HygroA-VL- (Gs4S) _ 3-VHHnpSecTag2 / HygroA-VH- (G4S) _ 3-VL and pSecTag2/HygroA-VL-p2A-VH,. Two cell lines Hela-VL-p2A-VH and CHO-VL- (G _ S) _ 3-VH.Western blot expressing A 尾 -specific scFv stably were obtained. The results showed that the corresponding scFv was expressed correctly. Indirect ELISA and dot blot showed that the secreted supernatant had the ability to recognize A 尾 antigen. In vitro experiments showed that it could block and inhibit the cytotoxicity of A 尾 oligomer. The stable expression of A 尾-specific single chain antibody cell lines is helpful for the further development of the basic research of AD immunotherapy.
【作者单位】： 北京交通大学理学院生命科学与生物工程研究院;中国疾病预防控制中心病毒病预防控制所;
【基金】：国家自然科学基金(81100809、81271417) 北京市自然科学基金(7152090) 中央高校基本科研业务费(2015JBM096)资助项目
【分类号】：R749.16
[Abstract]:Mammalian cell lines expressing A 尾 -specific scFv (scFv) were constructed. Using the method of overlapping extension PCR, using the light and heavy chain variable region of A 尾 -specific monoclonal antibody (A8) as template, the gene fragment of scFv was constructed by (GSP) _ 3 or p2A (Linker) sequence. A variety of scFv gene fragments were obtained by splicing to construct eukaryotic expression vector. The expression of scFv in human cervical cancer cell line (Hela) and Chinese hamster ovary cell line (CHO), Western blot was identified by liposome transfection. ScFv cell lines with stable expression of A 尾 were obtained by hygromycin screening, the antigen recognition ability of scFv was analyzed by indirect ELISA and dot blot analysis, and the cytoprotective effect of scFv was analyzed by cell experiment in vitro. Results: three eukaryotic expression vectors of A 尾 -specific scFv were successfully constructed: pSecTag2/HygroA-VL- (Gs4S) _ 3-VHHnpSecTag2 / HygroA-VH- (G4S) _ 3-VL and pSecTag2/HygroA-VL-p2A-VH,. Two cell lines Hela-VL-p2A-VH and CHO-VL- (G _ S) _ 3-VH.Western blot expressing A 尾 -specific scFv stably were obtained. The results showed that the corresponding scFv was expressed correctly. Indirect ELISA and dot blot showed that the secreted supernatant had the ability to recognize A 尾 antigen. In vitro experiments showed that it could block and inhibit the cytotoxicity of A 尾 oligomer. The stable expression of A 尾-specific single chain antibody cell lines is helpful for the further development of the basic research of AD immunotherapy.
【作者单位】： 北京交通大学理学院生命科学与生物工程研究院;中国疾病预防控制中心病毒病预防控制所;
【基金】：国家自然科学基金(81100809、81271417) 北京市自然科学基金(7152090) 中央高校基本科研业务费(2015JBM096)资助项目
【分类号】：R749.16
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