Construction Of CTB-PROIN And CTB-INSB Fusion Gene And Their Expression In Escherichia Coli And Transgenic Plant

Oral administration of disease-specific autoantigens can induce immunological tolerance, resulting to prevent or delay the onset of autoimmune diabetes symptoms. To develop an effective way for directly delivery of insulin to gut-associated lymphoid tissues, proinsulin (PROIN) and insulin B chain (INSE) gene were linked to the C-terminus of the cholera toxin B subunit (CTB), respectively. The pentameric CTB can be used as an efficient mucosal carrier of either immunogenic or tolerogenic T-cell epitopes. To investigate the possibility of producing vaccine for type 1 diabetes in potato, the two fusion genes, CTB-PROIN and CTB-INSB were transformed into potato plants. The main research and experimental results were summarized as follows.Firstly, the two genes, CTB-PROIN and CTB-INSB were cloned into pET-30a(+) to obtain a prokaryotic expression. The fusion proteins, which were expressed in E.coli, retained GM1-ganglioside binding affinity and native antigenicity of both CTB and insulin.Secondly, two plant expression vectors were constructed, which contained potato tuber specific promoter (Patatin), the fusion gene, potato proteinase inhibitor II gene's 3' UTR sequence and NOS terminator. The transgenic potato plants were obtained by nuclear transformation with agrobacterium mediated method. The fusion proteins synthesized by transgenic potato plants retained native antigenicity of both CTB and insulin, which can bind to GM1-ganglioside directly and possess potential biological activity. These results above encourage us to further analysize the immunogenicity of the fusion proteins.