Recombinant pBluescript II SK+ Vectors Harboring Human Thrombopoietin Gene

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. 본 발명은 인간 트롬보포이에틴 전체 또는 일부를 코딩하는 cDNA를 소의 β-카제인 유전자의 프로모터와 소의 성장호르몬 유전자의 폴리에이 첨가 신호서열(poly[A] signal sequence) 사이에 연결시키고 이를 플라스미드 pBluescript II SK에 삽입한 발현벡터에 관한 것이다. 본 발명에 의한 인간 트롬보포이에틴 cDNA를 함유하는 재조합벡터는 천연 인간 트롬보포이에틴과 유사한 분자량(74kDa)이며, 천연 인간 트롬보포이에틴과 동일하게 생체내/외 혈액세포-혈소판 증식효과를 나타내는 재조합 인간 트롬보포이에틴을 고농도(1500 ㎍/㎖)로 동물 생체반응기의 유즙에서 얻을 수 있게 함으로써 저렴하게 혈소판 감소증 치료제를 생산할 수 있게 된다.