Expression And Targeted Transportation Of Fluorescent Protein And T-PA-△EGF Fusion Gene In Chicken

Human tissue-type plasminogen activator(t-PA) is widely used in clinical treatment of cardiovascular diseases. T-PA is an effective thrombolytic without causing diffuse bleeding because of systemic fibrinolysis, it was the ideal drug to prevention and treatment of thrombosis. Apolipoprotein (apo-B100) is a necessary factor for the formation and assembles of very low density lipoprotein Y (VLDLy). During the course of biomacromolecules deposition in yolk, VLDLy was swallowed into growth yolk oocytes in the form of complete lipoprotein particles. To determine the feasibility about the accumulation of t-PA in the egg yolk and develop the functional eggs for prevention and therapy cardiovascular disease, the expression vector was constructed by means of fuse the functional t-PA fragment and apo-B100 gene. The recombinant plasmid pEGFP-N1-apo-tPA containing human t-PA and apo-B100 as well as green fluorescent fusion protein (GFP) fusion gene was constructed. When the resultant construct expression plasmid pEGFP-N1-apo-tPA was transformed into attenuated Salmonella typhimurium strain using electroporation method, the attenuated Salmonella typhimurium strain which carrying pEGFP-N1-apo-tPA was transfected henes by wing intravenous injection. The expression of fusion gene was detected by SDS-PAGE. The purpose of this study was to build a basis for gene transfer with the attenuated Salmonella and t-PA production from bioreactor of poultry.We have designed a pair of primers with molecular biology method, amplified fragment apo-tPA from the expression vector pCDNA3-apo-tPA, and inserted into pEGFP-N1 vector, when the resultant construct expression plasmid pEGFP-N1-apo-tPA was transfected into the fibroblast,the expression level of fusing protein was detected by ELISA, the biological activity of the expression product was detected by chromognic substrate methods, and then the expression plasmid pEGFP-N1-apo-tPA was transformed into attenuated Salmonella typhimurium strain using electroporation method, the attenuated Salmonella typhimurium strain which carrying pEGFP-N1-apo- tPA was transfected henes by wing intravenous injection. The results showed that the t-PA can be expressed in cultured chick embryo fibrocyte cells and secreted out cellular. The level of the highest expression was 577.6 ng/106 cells/24h and the expression product activity reached 148.3IU/106cells/24h in 48h. There was a target protein band of yolk protein at 1.06×105Da by SDS-PAGE electrophoresis. The attenuated Salmonella typhimurium were mainly distribute in liver, spleen and duodenum. The results of smear fluorescence microscopy and ELISA showed that there was t-PA accumulated in yolk on 8th day after the injection of attenuated Salmonella typhimurium, and the expression yield was 10.4μg/mL on 21th day; Fibrinolysis test showed that the activity of apo-tPA-GFP contained in yolk per milliliter corresponded to 50.2μg standard t-PA, since then has been decreasing slowly.The studies showed that apo-tPA-GFP fusion gene could be expression in vivo by attenuated Salmonella typhimurium and the expression product could deposition in yolk. It provides an effectual pathway for more proteins expressed in vivo. The fact that fusion t-PA gene mediated by apo-B100 expressed successfully in chicken demonstrated that the feasibility of the expressed foreign genes expressed in vivo and accumulated in the yolk. This approach processes the perspective in producing functional food which can be used to prevent the cardiovascular disease.