Research On Expression Of Recombinant Human Proinsulin In Arabidopsis

Diabetes mellitus (DM) is a chronic and systemic metabolic disease, which is characterized with hyperglycemia caused by absolute or relative scarcity of insulin. Therefore, insulin is the most effective drug to cure diabetes. The traditional insulin production via animal tissue extraction and microbial fermentation has several shortcomings. In recent years, plant bioreactor has been found to have several advantages on insulin production, yet some problems have been detected, such as excessive by-products and difficult purification. In this article, we considered producing modified recombinant human proinsulin in the model plant, Arabidopsis thaliana, via plant oil body expression system. The specific work included the following.1. Cloning of oleosin promoter and oleosin gene from huyou 15 (Brassica nape cv. Huyou 15). The human proinsulin gene (256 bp) was redesigned and synthesized according to plant codon usage preference. The proinsulin gene (mins) was connected to the downstream of oleosin gene (OG). A his-tag including 6 histidine residues was inserted in front of mins for the convenient detection and purification. The final oil body specific plant binary expression vector pCAMBIA2300-op::og-hmins (ohmins) was successfully constructed.2. Arabidopsis thaliana ecotype Colombia was transformed with A. tumefaciens GV3101 according to the floral dip method. The PCR results showed that 12 independent transgenic Arabidopsis plants were obtained among 15 resistant Arabidopsis regenerated plants which were screened by kanamycin (100 mg·L-1) with the transformed seeds.3. Southern blot analysis confirmed that 5 independent transgenic Arabidopsis plants were obtained. The ohmins gene was introduced into the genome of transgenic Arabidopsis plants, ranging from 1 to 4 copies.4. Real-time quantitative PCR results indicated that the target gene (ohmins) was transcribed in transgenic line.5. SDS-PAGE and Western blot results confirmed the expression of target protein in Arabidopsis seeds.6. ELISA analysis confirmed that the protein expression of the target gene ohmins accounted for 0.01～0.04% of total soluble protein (TSP) in Arabidopsis seeds.In this study, the plant binary expression vector was successfully constructed and independent transgenic Arabidopsis plants were obtained. Ohmins gene had been successfully expressed both at transcriptional and translational level in transgenic Arabidopsis. The study layed a good foundation for the future research on insulin expression through plant oil body expression system.