关键词: 转基因兔, 人组织纤溶酶原激活剂（ht-PA）, 乳腺特异性表达

Abstract: Human tissue-type plasminogen activator（ht-PA）is a major biomedicine used in the clinical treatment of thrombotic diseases. There have been some reports on transgenic animal mammary gland as bioreactor producing ht-PA, however, there has been no report about recombinant human-tissue plasminogen activator（rhPA）expressed in transgenic animals. Here we report the preparation of transgenic rabbits by microinjection of fertilized eggs with the constructed mammary gland-specifically expressed vector of PCL25/rhPA while using F, E, and K₁ loci deletants of human t-PA as coding sequence driven by the goat beta-casein/CMV chimeric promoter. PCR and sequencing PCR products were used to identify the transgenic rabbits from candidates, and ELISA and fibrin agarose plate assay（FAPA）to analyze the expressed products of the transgenic rabbits. Results showed that 5 rabbits（2♂, 3♀）were transgenic rabbits and rhPA were expressed in their milk of 2 rabbits with maximum content of 400 μg/mL, and its biological activity was 150-200 times of alteplase. This study implies that rhPA can be expressed specifically in the mammary glands under the regulation of goat beta-casein/CMV chimeric promoter, and the expressed recombinant protein possesses the high activity of thrombolysis.

Key words: transgene rabbit, human tissue-type plasminogen activator（ht-PA）, mammary gland-specifically expression