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

doi:10.13523/j.cb.20170201

China Biotechnology

2017, Vol. 37

Issue (2): 1-7 DOI: 10.13523/j.cb.20170201

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

WEN Jie¹, SONG Lin-lin¹, ZHANG Ying¹, WANG He¹, HE Jin-sheng¹, HONG Tao^1,2

1. College of Life Sciences and Bioengineering, School of Science, Beijing Jiaotong University, Beijing 100044, China;
2. Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China

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Abstract

The aim is to construct the stable mammalian cell lines which express the Aβ-specific single-chain 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 Aβ specifically, 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.

Key words： Alzheimer's disease (AD) Amyloid β-peptide (Aβ) Single chain fragment variant (scFv)

Received: 05 September 2016 Published: 25 February 2017

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Cite this article:

WEN Jie, SONG Lin-lin, ZHANG Ying, WANG He, HE Jin-sheng, HONG Tao. Construction and Function of Stable Mammalian Cell Lines Expressing the Aβ-specific Single Chain Fragment Variants. China Biotechnology, 2017, 37(2): 1-7.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170201 OR https://manu60.magtech.com.cn/biotech/Y2017/V37/I2/1

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