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  • Title: [Expression of human anti-HBsAg single chain Fab gene in Pichia pastoris].
    Author: Deng N, Xiang JJ, Su KY, Wang H, Tang Y.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2006 Jan; 22(1):71-3. PubMed ID: 16388750.
    Abstract:
    AIM: To investigate the relation of the affinity activity to the structure of recombinant Fab. METHODS: The human anti-HBsAg single chain Fab gene was obtained by overlaping PCR from the H and L chains of Fab, and was introduced into the expression system of Pichia pastoris. The expression vector of single chain Fab was transformed into GS115 cells by the method of LiCl transformation. The transformants were obtained and cultured in shake flask. The culture supernatant of the recombinant yeast was deposited in (NH(4))(2)SO(4) and purified by affinity chromatography. The affinity of culture supernatant of recombinant yeast and purified single chain Fab was analyzed by ELISA. RESULTS: The analysis of SDS-PAGE and Western blot showed that the single chain Fab gene was expressed successfully in Pichia pastoris, and the expression quantity was about 5-10 mg/L in shake flask. The recombinant Fab was purified by affinity chromatography and the purity reached 97.8%. The ELISA analysis showed that the recombinant single chain Fab had good HBsAg binding properties. CONCLUSION: The single chain Fab gene could be expressed successfully in Pichia pastoris and the recombinant single chain Fab can efficiently bind with HBsAg.
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