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

Construction and Function of Stable Mammalian Cell Lines Expressing the Aβ-specific Single Chain Fragment Variants

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。

The aim is to construct the stable mammalian cell lines which express the Aβ-specific singlechain fragment variants（ scFv）. In order to construct the scFv gene,the variable regions of light and heavy chain from Aβ-specific monoclonal antibody（A8） were used as the template, and splicing overlap extension polymerase chain reaction（ SOE-PCR） was used to get the scFv DNA fragments. Short sequence（G4S）3 or p2A was selected as the linker for scFv construction. The eukaryotic expression vectors for anti-Aβ scFv was constructed. Hela and CHO cells were transfected via Lipofectamine 2000,and the scFv products were detected through Western blot. The stable cell lines were screened with hygromycin B,and the antigen-binding ability of scFv in supernatants was detected by indirect ELISA and dot blot. The cellular protection ability of scFv was identified by cell transmission electron microscopy（ TEM）. Three eukaryotic expression vectors,pSecTag2 /HygroA-VL-（G4S）3-VH, pSecTag2 / HygroA-VH-（G4S）3-VL and pSecTag2 / HygroA-VL-p2A-VH were constructed; and two cell lines expressing the Aβ-specific scFv,Hela-VL-p2A-VH and CHO-VL-（G4S）3-VH were established. The results of indirect ELISA and dot blot showed that the supernatants could recognize Aβspecifically,and the results of cell assays in vitro showed that the supernatants could block the cytotoxicicity induced by Aβ oligomers. The stable cell lines expressing anti-Aβ scFv could be used for further AD immunotherapeutic study.