ABSTRACT
We have produced the recombinant intracellular domain of human IA-2 (IA-2ic), a diabetes-associated autoantigen, in plants. This was achieved by transient expression using agroinfiltration of Nicotiana benthamiana plants. The resulting plant-derived IA-2ic had the expected size, reacted with polyclonal and monoclonal antibodies specific to human IA-2ic and competitively inhibited radiolabeled IA-2ic in an immunoprecipitation assay. The expression level of recombinant IA-2ic was estimated to be 0.5% of the total soluble protein (TSP). Transient expression in plants has the potential to produce a large amount of human IA-2ic protein at low cost in a short period of time.
Subject(s)
Autoantigens/genetics , Autoantigens/immunology , Membrane Proteins/genetics , Membrane Proteins/immunology , Nicotiana/genetics , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/immunology , Antibodies, Monoclonal , Autoantibodies , Autoantigens/chemistry , Base Sequence , DNA Primers/genetics , Diabetes Mellitus, Type 1/immunology , Gene Expression , Humans , In Vitro Techniques , Membrane Proteins/chemistry , Plants, Genetically Modified , Protein Engineering , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/chemistry , Receptor-Like Protein Tyrosine Phosphatases, Class 8 , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
Historically, most vaccines have been based on killed or live-attenuated infectious agents. Although very successful at immunizing populations against disease, both approaches raise safety concerns and often have limited production capacity. This has resulted in increased emphasis on the development of subunit vaccines. Several recombinant systems have been considered for subunit vaccine manufacture, including plants, which offer advantages both in cost and in scale of production. We have developed a plant expression system utilizing a 'launch vector', which combines the advantageous features of standard agrobacterial binary plasmids and plant viral vectors, to achieve high-level target antigen expression in plants. As an additional feature, to aid in target expression, stability and purification, we have engineered a thermostable carrier molecule to which antigens are fused. We have applied this launch vector/carrier system to engineer and express target antigens from various pathogens, including, influenza A/Vietnam/04 (H5N1) virus.