بیان و هدفگیری انسولین انسانی در گیاه با استفاده از یک سامانه بیانی مبتنی بر حامل ویروس گیاهی

A plant viral vector engineered from an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) was used to express the human insulin in planta. The proinsulin gene was in frame inserted between the P1 and HC-Pro genes of the ZYMV vector. Six histidine residues and a NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate detection and process of free form of the expressed insulin, respectively. Also, N-terminal chloroplast signal peptide and C-terminal SEKDEL sequence was used to target insulin into chloroplast and endoplasmic reticulum (ER), respectively. The infectious activity of the recombinant vectors was approved by rubbing the plasmid on Chenopodium quinoa mechanically and observing local lesions. A single local lesion was inoculated on zucchini squash leaves and these plants were sampled 5, 10, 15 and 20 days after inoculation. Analysis of inoculated plants by enzyme-linked immunosorbent assay (ELISA) and western blot indicated that insulin protein was expressed in Squash leaves. RT-PCR result showed the recombinant vector was remarkably stable in squash for seven serial passages and after 30 days. Also, targeting of proinsulin using SEKDEL retention signal showed that targeting of the proinsulin to ER increased expression level of recombinant protein significantly. This procedure provides a convenient and fast way for production of large quantities of insulin in planta. This system has a potential for production of other proteins of interest in cucurbits to immunogen production, pharmaceutical applications, study on gene function, mass production of animal feed or use for other purposes.