Cloning and Expression of Interferon Beta-1a in CHO cell line

Cloning and Expression of Interferon Beta-1a in CHO cell line

Introduction: Interferon Beta-1a is a glycosylated protein, widely used as the first-line treatment of Relapsing – Remitting MS multiple sclerosis (RRMS). Expressed protein in E. coli as a non-glycosylated form has the highest immunogenicity while the glycosylated form, produced in Chinese Hamster Ovary cells (CHO), has the least immunogenic properties. This study intends to produce Interferon Beta-1a in Iran with many new RRMS cases recently. Method: The coding sequence of interferon beta-1a was cloned into the pSecTag A vector and transformed to E. coli top10 bacteria. Cloning process was confirmed using double digestion with BamHI and HindIII restriction enzymes and DNA sequencing. In order to investigate the expression of secretional interferon beta-1a, CHO cells were transfected with the recombinant vector (pSecTag A-INF), using TurboFect transfection reagent (ThermoScientific, USA). The cellular supernatant was collected after 24, 48 and 72 hours to be analyzed by 15% SDS-PAGE and subsequent Western blotting using anti-His-tag antibody. Result The reliability of cloning process was confirmed by appearing a band approximately in 900 bps on 0.8% Agarose gel electrophorese and by DNA sequencing. Supernatant analysis showed a band approximately in 24 KDa in 15% SDS-PAGE after 24 hours of transfection. The accuracy of expression was confirmed by Western blot analysis which revealed a band in 24 KDa. Conclusion We could successfully design and produce interferon beta-1a with potentiate ability of reducing MS symptoms. The expressed protein will be purified and the expression condition will be more optimized. However, it is essential to check its biological activities in the future studies.