Production of functional recombinant cyclic citrullinated peptide monoclonal antibody in transgenic rice cell suspension culture

• Published in: Transgenic research 2019-04, Vol.28 (2), p.177-188
• Main Authors: Van Giap, Do, Jung, Jae-Wan, Kim, Nan-Sun
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Agrobacterium tumefaciens - genetics; Animal Genetics and Genomics; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - metabolism; Antigens; Biological activity; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Cell culture; Cell Culture Techniques; Cells, Cultured; Chromosomes; Citrulline; Diagnosis; DNA-directed DNA polymerase; Enzyme-linked immunosorbent assay; Expression vectors; Genetic Engineering; Genetic transformation; Genetic Vectors; Humans; Life Sciences; Molecular Medicine; Monoclonal antibodies; mRNA; Original Paper; Oryza - genetics; Oryza - immunology; Oryza - metabolism; Oryza sativa; Peptides; Peptides, Cyclic - genetics; Peptides, Cyclic - immunology; Peptides, Cyclic - metabolism; Plant Genetics and Genomics; Plants, Genetically Modified - genetics; Plants, Genetically Modified - immunology; Plants, Genetically Modified - metabolism; Polymerase chain reaction; Protein G; Rheumatoid arthritis; Rice; Suspension culture; Transgenics; α-Amylase
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-019-00113-w

Cyclic citrullinated peptide (CCP) antibody has been shown recently to be a promising marker for early detection and diagnosis of rheumatoid arthritis (RA). In order to exploit newly developed therapies for RA, early intervention is crucial in preventing irreversible joint damage. Here, we describe use of a plant expression system to produce a CCP antibody that could be used in the early diagnosis of RA. Heavy and light chain gene sequences of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant expression vectors under the control of the rice α-amylase 3D ( RAmy3D ) promoter system. The vectors were introduced into rice calli ( Oryza sativa L. cv. Dongjin) using Agrobacterium tumefaciens mediated transformation. Integration of the CCP mAb genes into rice chromosomes was confirmed by a genomic DNA polymerase chain reaction and expression was verified by northern blot analysis of mRNA. The in vivo assembly and secretion of CCP mAb occurred in transgenic rice cell suspension culture under the RAmy3D expression system; accumulated CCP mAbs in the medium were purified by protein G affinity chromatography. Immunoblot assays and ELISA showed these plant-produced CCP mAbs successfully bound to a synthetic CCP antigen. Taken together, our results suggest that CCP mAb produced in a transgenic rice suspension culture were easily purified and biologically active against their antigen in the RA, and thus may be used a specific serological marker, which is present very early in the RA.