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Construction of a naive human phage scFv library and selection, identification of human scFvs

Qing Yuan1, Siji Nian1*, Xiyuan Guo1, Yan Gao1 and Jiajia Liu1
  • 1 Luzhou medcal college, department of Inmmunology, China

The IL-4, IL-13 and IL-33 are key components in the development of allergic disease such as asthma. To construction of human non-immune library and selection of human scFvs against the three cytokines, the mRNA of human peripheral blood cells (PBMC) was extracted from healthy volunteer, and then the cDNA library was constructed. The variable region of heavy chain (VH) and light chain (VL, including Vκ and Vλ) were amplified from full length cDNA library. The VH and VL were ligated with linker and the DNA library of single-chain Fv fragments (scFvs) was developed. After transforming into E.coli TG1, the phage library was constructed. The antigen IL-4, IL-13 and IL-33 were cloned, expressed and identified. The scFv library was enriched for three rounds by phage display for selection of human scFvs against IL-4, IL-13 and IL-33. By PCR, forty two bands of VH, sixteen bands of Vκ and eighteen bands of Vλ were got. And the scFv library was constructed successfully with 2.5x108 development clones with full length scFv inserts. Through three rounds of phage display, 30% clones showed positive using ELISA for detection. Four to five scFvs with high affinity against IL-4, IL-13 and IL-33 respectively were screened, expressed, and identified by Biacore, western blot and sequencing. The results showed the size of proteins of identified scFvs were about 32 kDa and the sequences were correct. The human non-immune library was constructed and the scFvs specific to IL-4, IL-13 and IL-33 respectively were selected successfully.

Acknowledgements

This work was supported by the Natural Science Foundation of China (No. 31000415)
and the project of Science and Technology Department of Sichuan Province (No.2012JQ0018).

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Keywords: Human scFvs, phage display, IL-4, IL-13, IL-33

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Adaptive Immunity

Citation: Yuan Q, Nian S, Guo X, Gao Y and Liu J (2013). Construction of a naive human phage scFv library and selection, identification of human scFvs. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00521

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Received: 07 Jun 2013; Published Online: 22 Aug 2013.

* Correspondence: Mr. Siji Nian, Luzhou medcal college, department of Inmmunology, Luzhou, Sichuan province, 646000, China, sijinian@hotmail.com