| Tissue-type plasminogen activator (t-PA) is an important drug for the treatment of thromboembolic disease. Since the first time t-PA has been proved that it has the efficiency of thrombolysis in 1980,t-PA because of its high affinity to fibrin,low tendency of systemic bleeding and the advantages of a strong ability to thrombolysis has been widely used in various thrombosis disease treatment.However, t-PA has some shortages all the same,its half life is only 3～5 minutes.So when used as therapeutic agent in vivo,it must be given continuously in abundance and the cost will be very high consequently.At present, the t-PA which is used for the clinical treatment of thrombosis disease depend largely on the production from mammalian cells, which exist defects of highly cost,small output , low concentration. As a result,the costs of t-PA in clinical treatment will be very high.Producing t-PA by animal mammary gland bioreactor not only maintain the low cost of production, but also highly yielded and can be translated correctly and modified.In this research,the mammary gland-specific vector pBCrt-PA was constructed.The F,E,K1 domain and the glycosylation site Asp 117 were deleted from the cDNA of wild human t-PA. The modified cDNA and the regulatory sequence of goatβ-lactoglobulingene were assembled into mammary gland-specific construct. Then, pBCrt-PA was transfected into goat mammary epithelial cells cultured in vitro and the expression cell strains were obtained.Firstly,human t-PA variant cDNA was cloned from wild t-PA cDNA,which was 1.1kb and comprised only K2 and P domain coding regions.At the same time,the goatβ-lactoglobulin gene signal peptide coding sequence was synthesized in vitro,which was 60bp.By the strategy of directional cloning,the K2 and P domain coding regions and goatβ-lactoglobulin gene signal peptide coding sequence was fused with the commonly primary mammary gland–specifice vector BnF95.At last,the mammary gland -specific vector pBCrt-PA for a variant of t-PA was constructed.Further,the plasmid pBCrt-PA was digested by Sal I and Not I at the same time the 9.8kb transgenic fragment was extracted. By electroporation, the transgenic fragmen was transfected into goat mammary epithelial cells line in vitro culture. After selection of three weeks by G418, the stable transfected cell clonies were obtained. There were clonies after the individual healthy clonies, which was picked into another tissue culture dish to continue culturing. DNA sample of these 62 clonies were screened by PCR and 22 clonies had been successfully transfeced with foreign genes. These clonies were induced with prolactin and then the liquid of inducing was gleaned at 48h. It was found that there were 2 clonies expressing t-PA variant by ELISA detected at 48h. But the expression level of t-PA variant was relatively low and their expression level of t-PA variant were about 5.98ng/ml and 2.35ng/ml respectively.Thus,our subsequent work should focus on improving expression level of this t-PA variant and further study for the bioactivity and half life of expression product.The ideas of constructing the vector and t-PA variant gene cloning provid a powerful experimental basis and important guiding significance for the future of mading transgenic goat mammary gland bioreactor to mass produce the new thrombolytic drugs which has strong specific thrombolytic activity and long half-life.
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