Expression of recombinant chains of anti-VEGF monoclonal Ab in yeast

INTRODUCTION: Age-related macular degeneration (AMD) is the major cause of blindness in the elderly and vision loss in patients with diabetic retinopathy. This complication is usually associated with uncontrolled secretion of VEGF. Anti-VEGFs medications, with different generic names like Bevacizumab, Pegabtanib, Ranibizumab are the common treatment used to control the disease progression. OBJECTIVE: The aim of present study is to produce the two recombinant monoclonal antibody chains in yeast Pichia pastoris and evaluate the whole antibody for interaction with VEGF.MATERIALS AND METHODS: The sequence of light and heavy chains of antibody were cloned in the pPinkα-HC vector in frame with the αMF secretion signal and under the AOX1 prometor control. After propagation in Escherichia coli, recombinant plasmids were incorporated into the genome of yeast strain GS115 and the positive clones confirmed by PCR and sequencing. The recombinant proteins of light and heavy chains were expressed by methanol induction, evaluated using SDS-PAGE and functionality of final structure investigated by ELISA using VEGF as substrate and HRP conjugated anti-Fab specific Ab for detection. RESULTS: two chains of antibody are produced in yeast and secreted in culture medium. After 24 h of incubation of heavy and light chains, the whole structure effectively interact with VEGF.

کلیدواژه ها:

Monoclonal antibody ، Recombinant protein ، Pichia pastoris ، ELISA