쥐 프로트롬빈 cDNA 의 클로닝과 발현

Abstract

A rat liver cDNA library was screened for a prothrombin cDNA using a cDNA fragment coding human prothrombin as a probe. Among about 50,000 plaques, 30 positive plaques were obtained by hybridization with ^(32)P-labelled probe. The cDNA insert from two of the positive λ DNA were selected for further analysis of the prothrombin cDNA. The DNA sequence of the cDNA coding a rat prothrombin has been determined. It consists of 1950 nucleotides and encodes an ORF of 1851 nucleotides, 22 bp noncoding regions at 5 end and 97 bp noncoding regions at 3 end. It does not have poly A tail and poly A addition signal, AATAAA. Our cloned prothrombin cDNA differs by 6 nucleotides from already cloned rat prothrombin cDNA. It shows 80% homology with human prothrombin. Comparative analysis of region having considerable sequence homology between human and rat prothrombin suggests that the functional and structural domains and post translational modification sites in rat prothrombin cDNA can be mapped. The signal peptide, the cleavage sites, γ-carboxylated region, glycosylation attachment sites and the cleavage sites by Factor Xa in rat prothrombin can be presumed by comparative analysis. The rat prothrombin cDNA was expressed in E. coli as a fusion protein using glutathione S-transferase vector system.