【摘要】：構(gòu)建可以穩(wěn)定表達(dá)Aβ特異性單鏈抗體(scFv)的哺乳動(dòng)物細(xì)胞株。應(yīng)用重疊延伸PCR的方法,以前期建立的Aβ特異性單克隆抗體(A8)的輕、重鏈可變區(qū)基因?yàn)槟０?構(gòu)建scFv的基因片段,通過(G_4S)_3或p2A兩種不同的連接肽(Linker)序列,拼接得到多種形式的scFv基因片段,用于構(gòu)建真核表達(dá)載體。利用脂質(zhì)體分別轉(zhuǎn)染人宮頸癌細(xì)胞(Hela)和中國倉鼠卵巢細(xì)胞(CHO),Western blot鑒定scFv的表達(dá)情況;以潮霉素篩選獲得穩(wěn)定表達(dá)抗Aβ的scFv細(xì)胞株,以間接ELISA和斑點(diǎn)印跡分析所得scFv的抗原識別能力;采用體外細(xì)胞實(shí)驗(yàn),在超微病理水平分析所得scFv的細(xì)胞保護(hù)作用。結(jié)果:成功構(gòu)建了Aβ特異性scFv的3個(gè)真核表達(dá)載體pSecTag2/HygroA-VL-(G_4S)_3-VH、pSecTag2/HygroA-VH-(G_4S)_3-VL和pSecTag2/HygroA-VL-p2A-VH,獲得了2株穩(wěn)定表達(dá)Aβ特異性scFv的細(xì)胞株Hela-VL-p2A-VH和CHO-VL-(G_4S)_3-VH。Western blot結(jié)果表明了相應(yīng)scFv的正確表達(dá),間接ELISA和斑點(diǎn)印跡結(jié)果表明所分泌的細(xì)胞上清具有Aβ抗原識別能力,體外實(shí)驗(yàn)顯示其具有阻斷和抑制Aβ寡聚體細(xì)胞毒性的作用。穩(wěn)定表達(dá)Aβ特異性單鏈抗體的細(xì)胞株有助于AD免疫治療基礎(chǔ)研究的進(jìn)一步開展。
[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.
【作者單位】： 北京交通大學(xué)理學(xué)院生命科學(xué)與生物工程研究院;中國疾病預(yù)防控制中心病毒病預(yù)防控制所;
【基金】：國家自然科學(xué)基金(81100809、81271417) 北京市自然科學(xué)基金(7152090) 中央高�；究蒲袠I(yè)務(wù)費(fèi)(2015JBM096)資助項(xiàng)目
【分類號】：R749.16

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