Increased Expression Of The Truncated Human Tissue Plasminogen Activator (Reteplase) Using A Plant Virus-based Expression System

Reteplase is the representation medicine used in therapy for the thrombotic diseases in clinic. For now, commercial Reteplase is costs high in production and expense, to solve this problem is to explore and figure out the new and efficient express system for improving the express production. In the laboratory, we used to try to use TMV express Reteplase in N. benthamiana expression system and HFBI expression technique, while the outcome is low with this method.It showed that the TMV expression system and speculate that the replacement of signal peptide and codon optimization are the possible cause of the low outcome. In view of this, this research aims to definite the expression impact of the recombination Reteplase in botany by different source of signal peptide and different codon optimization, meantime, combining the feature of RNA silence suppressors coded by virus, optimizing the Reteplase expression system and figure out the expression rule changed followed by time, we also established the purification method of Reteplase and analyzed the physicochemical and biological characteristics of the purified Reteplase.In the process of optimization in the expression system, we firstly used the bioinformatics software named SignalP 4.1 to evaluate the signal peptide from different source. We designed and built two Reteplase expression systems through the gene synthesis and subcloning technology. Then the host botany (N. benthamiana) was infected by Agrobacterium infiltration method and the expression efficient of the recombination Reteplase was infected. As the result we got, the expression efficient of Reteplase mediated by the Pr1b signal peptide from botany source is much higher than the "nature" signal peptide mediate. Secondly, we used GeneOptimizer(?) to optimize the codon of Reteplase encoding genes based on the frequency of use of the N. benthamiana codon and N. tabacum codon, we used the NetGene2 to reevaluate the gene sequence which had already optimized the codon. On the basis of results above, we built three different virus expression systems of Reteplase. The results of the analysis after inoculated botany showed that in different infection concentration, the expression system which optimized sequence is more efficient than the expression system which sequence did not be optimized. Above this, we could draw the conclusion that the optimization of codon is the indispensable step of improve the expression efficient. Thirdly, co-infected with HC-Pro which is the RNA Silencing Suppressors (RSSs) coded by Tobacco etch virus (TEV) can improve the expression of Reteplase in botany. The max expression quantity is about 12.5μg in per gram fresh leaves and the best collect time is about the 9th day in the Reteplase expression system. The conclusion we have got is that it is feasible to use the botany as the bio-reactor to express the Reteplase. Fourthly, we found that it may necrosis in the part of leaves infected in the Reteplase expression virus but not occurred in the empty agrobacterium group and GFP group. What’s important was that the order of severity of necrosis is positive correlation with the expression of Reteplase. So we speculated that the recombination Reteplase may have the cytotoxicity to N. benthamiana botany cells and the cytotoxicity is related to the expressive quantity of Reteplase.During purification of the Reteplase by Strep Ⅱ tag technology, we found that expression quantity of recombination Reteplase in endoplasmic reticulum is higher than out of the cells. In addition, the location of Strep Ⅱ tag is also impact the expression efficient of recombination Reteplase. When the Reteplase was purified, the Avdin was added into combining buffer, it could efficiently inhibit the impact of the purification by plant endogenous biotin, thus it could improve the binding efficiency between Reteplase and affinity matrix. There were several bands through SDS-PAGE and Western blot to detect the purification Reteplase, we analyzed and found that the bands were the product of the self-protease cleavage site broken of recombination Reteplase.The physicochemical properties of the purificated Reteplase showed that the molecular weight of purificated Reteplase is 45.0 kD and it is slight greater than the standard sample which is non-glycosylation by prokaryotic expressed (39.0 kD). Glycosylation experiment shows that the difference of the molecular weight is due to the Reteplase have two glycosylation sites. The in vitro biological activities experiments revealed that the purification Reteplase could produce the obvious solusphere phenomenon. Above all, we can get the conclusion that the recombination Reteplase expressed in N. benthamiana have the biology activity and the activation is as the same as the commercialization Reteplase expressed by prokaryotic expression system. It could also be proved that it is viability that used botany virus vector expression system to express Reteplase in N. benthamiana botany. Meanwhile, a foundation was established to improve the expression of recombination Reteplase in botany expression system.