The Effect Of The Necrosis On Reteplase Expression In Tobaceo Leaves

Reteplase is the third-generation thrombolytic drug commonly used in therapy for the thrombotic diseases in clinic.It is great significance to explore and study a new and efficient expression system for the clinical application of Reteplase.In the early stage of laboratory,we tried to express the recombinant Reteplasein in the endoplasmic reticulum of N.benthaminana by using the vector expression system of Tobacco Mosaic virus(TMV).After the expression of recombinant Reteplase,the phenomenon of cell necrosis in leaves was observed.The reason for this is not yet clear.Considering the universality of this phenomenon,cell necrosis has a certain degree of influence on the quantification and downstream purification of expression products.Therefore,the main factors of analyzing the phenomenon have certain practical significance for the rational use of plant virus vector expression system.Based on this,we carried out related research from the analysis of the main factors of necrosis symptoms,measures to improve the symptoms of necrosis,and the use of protective label(Zera)to express recombinant Reteplase.In order to explore the main factors of necrotic symptoms,the study was carried out from two aspects,replacement of exogenous genes and replacement of viral expression vectors system under the same experimental conditions.Firstly,based on the expression vector of pTMV rPA-NSK virus,the TMV expression vector pTMV GFP-NSK was constructed by replacing the Reteplase coding gene.The phenotypic and exogenous gene expression was compared with that of pTMV rPA-NSK vector after inoculation of the recipient plant.The results showed that the pTMV GFP-NSK vector expressed the reporter gene GFP and no necrosis was observed throughout the observation period.It was preliminarily confirmed that the occurrence of necrotic symptoms was not caused by the TMV virus vector alone.Secondly,the TMV viral expression vector was replaced with the CPMV viral vector to construct the CPMV rPA-NSK vector.The results showed that the CPMV rPA-NSK viral expression vector could express recombinant Reteplase without causing symptoms of necrosis.Although,TMV viral vector expression is slightly higher than CPMV viral vector expression.However,the expression of CPMV virus vector on the 7th and 9th day after inoculation was higher than the expression of TMV virus vector on the 5th day after inoculation.The TMV virus expression vector produced significant necrosis on the 5th day after inoculation.The CPMV viral vector expression could not observe necrosis on the 9th day after inoculation.Therefore,it was considered that the symptoms of leaf necrosis after expression were not caused by recombinant Reteplase alone.Based on the above experimental results,it was considered that the TMV viral vector and the recombinant Reteplase together caused the occurrence of necrosis symptoms.On the basis of identifying the main factors causing the necrosis of leaves after expression,we try to improve the necrotic symptoms both from the alteration of the expression space of the recombinant Reteplase cells and the replacement of the host plants.Firstly,based on the endoplasmic reticulum expressed pTMV rPA-NSK viral vectors,TMV viral expression vectors expressed in extracellular,mitochondrial and chloroplast were constructed.The results inoculation showed that there was no necrosis in the extracellularly expressed viral vectors,but no expression of recombinant Reteplase was detected in the samples.Severe necrotic symptoms were observed in the mitochondrial-expressed viral vectors,and only slight expression was detected.The virus vector expressed in chloroplast showed no necrosis,and the amount of expression was lower than that of recombinant Reteplase expressed in endoplasmic reticulum(ER).The virus carrier expressed in the endoplasmic reticulum appeared to be less severe than the mitochondria,and the recombinant Reteplase had the highest expression level.The results showed that there was a difference in the expression efficiency of cell expression space in Reteplase and in the phenotype and extent of cell necrosis.Secondly,the pTMV rPA-NSK virus vector was inoculated into two kinds of receptor plants,N.benthamiana and N.excelsiana,respectively,to examine the effect of the host plants on the expression.The results showed that the symptoms on N.benthamiana were more severe than N.excelsiana,while the expression of recombinant Reteplase in N.benthamiana was slightly higher than that of N.excelsiana.The results showed that there are differences in the degree of necrotic phenotype between different receptor plants.It is considered that N.excelsiana is more suitable as a TMV viral vector host plant expressing recombinant Reteplase.In order to solve the problem of internal breakage of the recombinant Reteplase,the research on expression of recombinant Reteplase using the Zera protective tag was carried out.The results showed that the formation of Zera taginduced particles in the plant cells was a dynamic process from soluble to aggregation.The aggregation of insoluble protein particles can protect the integrity of recombinant Reteplase to a certain extent,while the soluble form of the recombinant Zera-rPA product will still be attacked by the cellular protease,resulting in a decrease in the effective protection rate of the protective label.