Production of functional recombinant cyclic citrullinated peptide monoclonal antibody in transgenic rice cell suspension culture
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.
KeywordsCyclic citrullinated peptide monoclonal antibody (CCP mAb) Rice α-amylase 3D promoter Transgenic rice cell suspension culture
This research was supported by the Advanced Production Technology Development Program, Ministry for Food Agriculture, Forestry and Fisheries (312037-05) and Do Van Giap was supported by the BK21 plus program in the Department of Bioactive Material Sciences, Chonbuk National University, Republic of Korea. The authors are appreciative of Dr. JH Ju of Catholic University for providing hybridoma cell line and also grateful to Dr. YS Jang of Chonbuk National University for performing the cloning of heavy and light chain of CCP from hybridoma cell.
DV and JW carried out almost all experiments in this study and prepared the manuscripts. NS provided suggestions for experiments and wrote the manuscript. All the authors have read and approved the final manuscript.
Compliance with ethical standards
The authors declare that they have no competing interests.
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