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Expression of recombinant Hirudin in transgenic mice milk driven by the goat β‐casein promoter
Author(s) -
Yen ChonHo,
Yang ChiKai,
Chen IChung,
Lin YinShen,
Lin ChihSheng,
Chu Sen,
Tu ChingFu
Publication year - 2008
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200800069
Subject(s) - hirudin , recombinant dna , transgene , microbiology and biotechnology , thrombin , biology , biochemistry , casein , chemistry , gene , platelet , immunology
Hirudin, isolated from the leech Hirudo medicinalis, inhibits thrombin directly and several expression systems have been used to produce recombinant Hirudin (rHirudin) for pharmaceutical purposes. A DNA fragment containing the Hirudin coding sequence and goat β‐casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat β‐casein expression vector for mouse transgenesis. Four lines of transgenic mice were successfully developed and one line showed a meaningful anti‐thrombin activity of 40,000 anti‐thrombin units (ATU)/mL in their milk. In this animal line, Hirudin mRNA was found in samples of uterus and kidney with insignificant anti‐thrombin activity (≤ 280 ATU/g wet tissue); however, mammary glands showed a higher activity of 780 ATU/g wet tissue. Transgenic mice showed no evident physical abnormality. The purified rHirudin was further analyzed by amino acid analysis and was found to contain a tyrosine O ‐sulfate residue that is absent in rHirudin expression either through Escherichia coli or yeast host systems. Experimental results demonstrated that the β‐casein‐promoted Hirudin transgene could be successfully expressed in a murine model and may be applicable to large mammals such as livestock for mass production of rHirudin for pharmaceuticals.