Expression Of The Truncated Human Tissue Plasminogen Activator(Reteplase) In Plant Tobacco Based On Hydrophobin HFBI

The high morbidity and mortality of acute myocardial infarction, cerebral infarction and other thromboembolic diseases, threaten seriously to human health and life. Reteplase is a common drug for treatment of thrombotic diseases in the clinical. The current commercial Reteplase is achieved through E. coli expression system, which is non-glycosylation. The purity of production is low and expensive. Therefore, the key issues are to explore and study a new, simple and high expressive level in expression system. Our group has tried to express Reteplase in Tobacco by the Tobacco Mosaic virus RNA-based expression vectors. The results showed that it was feasible to utilize plants as the bioreactors to express Reteplase, while Reteplase expressed in Tobacco Mosaic Virus RNA-based expression vector system through Nicotiana benthamiana was degraded seriously. In this view, the possibility of Reteplase expressed in Tobacco through HFBI fusion would be explored and utilized.With GFP, the report gene, the effect and characteristics of HFBI fusion for expressing to the foreign genes were researched first. Through Agrobacterium infiltration technology, HFBI fusion GFP expression vector were respectively inoculated in host plant N. benthamiana and N. tabacun cv. White Burley. In comparison with the whole plant and cellular levels of tobacco vaccination site fluorescence signals from two alternative expression hosts, N. benthamiana was selected as HFBI fusion express foreign genes of host plants. During the experimental observation period, the experiments of whole plants, cells and molecular experiments indicated that fluorescence signal/expression quantity of GFP-HFBI was significantly higher than GFP in each period. It was proved that HFBI fusion expression could effectively improve the expression level of foreign genes in tobacco plants. Meanwhile through a comparative analysis of nucleic acid (mRNA) and protein levels of GFP and HFBI-GFP expression accumulation levels in different periods, we speculated that HFBI played a certain role in the translation level to improve the expression level of exogenous genes rather than in the transcription level in tobacco. Further observations of cellular level showed that a lot of dense green fluorescent protein particles of GFP could be seen in tobacco leaf cells in HFBI fusion expression, while it was not be observed this phenomenon in the control group. It was believed that HFBI fusion expressive products could forme insoluble aggregates in the cell because HFBI has hydrophobic and easy aggregation properties. Then in a certain extent, the expression products degradation through endogenous protease in the host cell was been isolated in order to achieve the long-standing and last accumulation of foreign genes’expressive products. By the additive effect, the expression level was improved in the exogenous genes. Based on the results above, the study on the fusion expression of Reteplase in tobacco was developed by using HFBI. By taken the codon of ordinary cigarette as the usage frequency, the gene encoding sequences of Reteplase were conducted for the codon optimization and gene synthesis, and the HFBI plant expressive vector for expressing Reteplase was constructed through sub-cloning technology. Through SDS-PAGE and Western blotting, it was showed that Reteplase could be expressed in N. benthamiana by HFBI fusion expression, the quantity is about5.0μg/g of fresh leaves. Results of Biological activity experiments in vitro showed that, Reteplase expressed in tobacco has fibrinolysis activity in vitro, and tag HFBI expressed in the N-terminal fusion expression had not a significant impact on the biological activity of it. Through experimental results above and prediction of bioinformatics, it was speculated that there are two possible reasons about high expressing GFP by HFBI fusion expression in tobacco (2.5mg/g fresh leaves) and lower expressing Reteplase (5.0μg/g fresh leaves). Firstly, a potential intron splice sites was produced because of the optimization of codon; Secondly, the original signal peptide sequence of Reteplase could not be correctly identified and cut by plants.Results in the dissertation are valuable and practical for further investigation on HFBI fusion expression and guidance to the expression of commercial exogenous genes. Meanwhile, there will be the support to construct a new-style and alternative expression system for Reteplase.