Expression of recombinant Hirudin in transgenic mice milk driven by the goat beta-casein promoter.
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 beta-casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat beta-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 beta-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.
Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan, R.O.C.
Chon-Ho
CH
Yen
Chi-Kai
CK
Yang
I-Chung
IC
Chen
Yin-Shen
YS
Lin
Chih-Sheng
CS
Lin
Sen
S
Chu
Ching-Fu
CF
Tu
D000818:Animals; D002364:Caseins / Q000235:genetics*; D005260:Female; D006041:Goats; D006629:Hirudins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D008892:Milk / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
D002364:Caseins; D006629:Hirudins; D011994:Recombinant Proteins