[Preparation and identification of single-chain fragment variable against epidermal growth factor receptor 3].

Autor:	Liu M; Guangdong Provincial Key Laboratory for Biotechnology Drug Candidates, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China., He P; Guangdong Provincial Key Laboratory for Biotechnology Drug Candidates, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China., Wen B; Guangdong Provincial Key Laboratory for Biotechnology Drug Candidates, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China., Qiu C; Guangdong Provincial Key Laboratory for Biotechnology Drug Candidates, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China., Li H; Guangdong Provincial Key Laboratory for Biotechnology Drug Candidates, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China., Zhao L; Guangdong Provincial Key Laboratory for Biotechnology Drug Candidates, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China. *Corresponding author, E-mail: 50907053@qq.com.
Jazyk:	čínština
Zdroj:	Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology [Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi] 2019 Oct; Vol. 35 (10), pp. 938-943.
Abstrakt:	Objective To express, purify and identify the single-chain fragment variable (scFv) against human epidermal growth factor receptor 3 (HER3). Methods We searched NCBI for the light chain sequence and heavy chain sequence of anti-HER3 mAb LJM716 to construct the gene of scFv against HER3. The recombinant expression vector pGAPZαA-anit-HER3-scFv was constructed using the constitutive expression vector pGAPZαA and then electro-transformed into Pichia Pastoris X-33 to screen the strains with high expression of the protein of interest. After shaking flask fermentation, the supernatant was purified by hydrophobic chromatography and metal ion affinity chromatography. The purified product was identified by Western blotting and ELISA. Results The anti-HER3-scFv gene was successfully constructed and the strains with high expression of anti-HER3-scFv were obtained. The anti-HER3-scFv was purified to a purity of more than 95% by two-step chromatography, and the purified yield was 192 mg/L. Western blotting showed that the anti-HER3-scFv was correctly expressed and ELISA indicated that anti-HER3-scFv could specifically recognize HER3. Conclusion The anti-HER3-scFv has been successfully prepared.
Databáze:	MEDLINE
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