Transient expression of bioactive recombinant human plasminogen activator in tobacco leaf / 南方医科大学学报

OBJECTIVE@#To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production.@*METHODS@#Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by sequence synthesis and subcloning. The two vectors were inoculated on either or leaves agroinfiltration. The expression of recombinant rhPA in leaves was examined using Western blotting and ELISA, and the fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA).@*RESULTS@#Five to nine days after infiltration with an inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated leaves was significantly higher than that in leaves ( < 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles.@*CONCLUSIONS@#Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.

A pilot study on purification of human plasminogen from cord blood plasma and inducing posterior vitreous detachment / 中华实验眼科杂志

Background Many studies and clinical trials of pharmacologic vitreolysis are already under way to try to improve vitreo-retinal surgery and to liquefy and detach the vitreous from the retina ultimately, including chondroitinase,hyaluronidase,dispase and plasmin. However, there has not been any report on purification of human plasminogen from cord blood plasma and inducing posterior vitreous detachment of the animal eye at present.Objective This study was designed to isolate and purify the production of human plasminogen (Plg) from cord blood plasma with ethanol precipitation and evaluate the efficacy of Plg in inducing posterior vitreous detachment (PVD).Methods Human Plg was Separated and purified from cord blood plasma by ethanol precipitation method. The protein band corresponding to Plg with molecular mass of 92 000 was revealed in SDS-PAGE and confirmed by MALDI-TOF and Mascot database. Anion-exchange chromatography and plasminogen activity assay kit were used to obtain purified Plg with biological activity. Twenty-five fresh pig eyes were enucleated and assigned to 5 groups and 5 eyes for each group. The normal eyes were used as control group. Balanced salt solution(BSS)of 0.1 ml was intravitreally group and standard substance group. All of the eyes were then incubatedfor 60 minutes under the 37 ℃. Retinal histopathology and ultrastructure were examined under the light microscopy, scanning electron microscopy ( SEM ) and transmission electron microscopy (TEM). Results The Plg with potential fibrinolytic activity was successfully extracted and purified from cord blood plasma by ethanol precipitation method. No posterior vitreous detachment (PVD) was seen in normal control group, BSS group and r-SK group following the intravitreal injection under the sem. However,PVD was demonstrated in r-SK+ Plg group and standard substance group under the SEM. The inner limiting membrane ( ILM ) and the retina were well preserved in all of the experimental eyes. No retinal morphology and ultrastructural abnormality were found under the light and SEM and TEM. Conclusion Ethanol precipitation is a feasible way to isolate and purify Plg from human cord blood plasma. Extracted Plg shows potential fibrinolytic intravitreal injection of Plg.

Construction and expression of eukaryotic expression vector for recombinant human plasminogen Kringle 5 gene / 上海第二医科大学学报

Objective To construct baculovirus expression system for the human plasminogen kringle 5(hK5) gene,and detect its expression in Spodoptera frugiperda 21(sf21). Methods Baculovirus Bacmid-rhK5 was prepared with bac-to-bac baculovirus expression system,and sf21 cells were infected.The expression of rhK5 in sf21 cells was determined by Western blot.The activity of purified protein was detected by vascular endothelial cell inhibition test. Results The pFastBac HTB-rhK5 was successfully constructed,and sf21 cells were transfected with the constructed vector.The output of rhK5 obtained was 90 ug/L,and the protein possessed the inhibitory activity of endothelial cell proliferation.The median effective dose(ED50) was 4 ug/mL. Conclusion The rhK5 baculovirus expression vector is highly expressed in sf21 cells,which lays a foundation for the the expression of rhK5 protein with biologic activities.