Abstract
The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2–630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.
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Acknowledgments
This study was supported by the National Major Special Projects on New Cultivation for Transgenic Organisms (2011ZX08008-004), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Jiangsu Province Science and Technology Support Project (BE2013679) and the Graduate Research and Innovation Projects in Yangzhou University (CXLX-1435). We thank our teacher, Professor Yong-Cheng, for providing technical assistance. We would also like to thank the laboratory personnel for their roles in helping with this study.
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Song, S., Ge, X., Cheng, Y. et al. High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro. Mol Biol Rep 43, 775–783 (2016). https://doi.org/10.1007/s11033-016-4020-0
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DOI: https://doi.org/10.1007/s11033-016-4020-0