Recombinant pBluescript II SK+ Vectors Harboring Human Thrombopoietin Gene

摘要

PURPOSE: A recombinant vector comprising cDNA which encodes human thrombopoietin(hTOP) is provided. Thereby, high concentration (15 microgram/ml) of recombinant hTOP is prepared which has a similar molecular weight of 74 kDa as natural hTOP and which shows the same proliferation effect of hemocyte-platelet as natural hTOP in vivo/in vitro. CONSTITUTION: The recombinant vectors comprising cDNA, which encodes whole human thrombopoietin(hTOP) of aminoacid sequences(1-353) or partial hTOP of aminoacid sequences (1-174), are prepared by the following steps of: performing PCR using hTOP as a template to prepare cDNAs of each 1061 bp and 520 bp in size; placing a cDNA at the SacII site of a bovine beta-casein gene promoter in down stream; ligating a DNA fragment of 300bp, which is the poly £A| signal sequence of a bovine growth hormone gene, at the Kpnl site of each cDNA in down stream; inserting and ligating a recombinant cDNA between Sacl and Notl sites of pBluescript II SK+ to obtain a expression vector. The recombinant vector is micro-injected into a mouse fertilized egg by conventional methods to obtain a transformed mouse then TOP RNA is expressed only in its mammary gland. hTOP for the therapeutic agent of thrombocytopenia can be mass produced from bovine milk in low cost by applying the recombinant vector into an animal bioreactor.