Expression And Activity Detection Of Apo-tPA Fusion Protein In Chicken And Its Accumμ Lation In Yolk

Human tissue-type plasminogen activator (t-PA), a specific, fibrin-selective thrombolytic agent was one of the most effective drugs to prevent and treat the thrombotic diseases. Apolipoprotein apo-B100 was necessary for the formation and assembles of very low density lipoprotein (VLDLy). During the course of deposition of organic matter in the yolk, VLDLy was swallowed into growth yolk oocytes in the form of complete lipoprotein particles. To determine the feasibility about the accum- ulation of t-PA in the egg yolk and develop the functional eggs for anti cardiovascular disease, the expression vector was constructed by means of fuse the fu-nctionalΔt-PA fragment in t-PA gene and apo-B100. After identification the correct expression fram- ework in the model of hepatocyte in vitro, the expression vector was transfected into the chicken by liposome. Which the study of the expression of the fusion protein in the chicken and deposition in the egg yolk pave an road on the use of poultry as a bior- eactor to produce t-PA.The total RNA of layer liver was extracted by molecular biology techniques in this study. The apo-B100 gene cloned using specific primers by RT-PCR was fused with theΔt-PA, functional area fragment t-PA via linker protein. It was cloned into downstream of CMV promoter of pCDNA3 expression vector and obtained pCDNA3-apo-ΔtPA expression vector. Then the expression vector was transfected into the hep- atocyte to analyze the correct reading frame. The expression level of fusing protein was detected by ELISA. The biological activity of the expression product was detected by chromognic substrate methods. The expression vector packaged with liposome was injected into chicken by intravenous injection. The egg was collected at different time. The expression product was detected by SDS protein electrophoresis and Western blotting; the level of expression was detected by ELISA; the biological activity of exp-ressed product was detected by chromognic substrate mothed; the expression and distribution of the fusing protein in different organizations organs in the Chicken were detected by immunohistochemisty.The results showed that the t-PA fused gene mediated by apo-B100 gene fragment of 1479bp which cloned by RT-PCR can be expressed in cultured chick embryo liver cells and secreted out cellular. The level of the highest expression was 675.3ng/106 cells/24h and the expression product activity reached 135.8IU/106cells/24h in 48h. There was a target protein band of yolk protein in 84×10~3 by SDS protein electroph- oresis and which was been proved corrected by Western blotting test; the level of the highest expression of the fused protein was 7.8mg/L 3w after injected with plasmid, then declined to 1.8mg/L on the 9w. And the fused protein was biological activity both in vivo and in vitro analysis. The distribution of the fused protein in the heart, liver, blood vessels, fallopian tube, muscle, kidney, and other organizations in the Chicken were detected by immunohistochemisty, the fused protein was higher expression in liver and muscle cells than other organization. The fact that fusion t-PA gene mediated by apo-B100 was can be expressed successfully in chicken demonstrated the feasi- bility of the expressed foreign genes to deposit in the yolk. And this approach proc- esses the perspective in producing functional food which can be used to prevent the cardiovascular disease.