| The vegetable cheese Natto is a typical and popular soybean fermented food, having been utilized for various purposes in folk medicine more than 2000 years in Japan. In 1988, Sumi et al found a strong fibrinolytic enzyme, named Nattokinase (NK), in Natto and examined some of its properties. In vivo, the Natto-saline extract on normal human volunteers by oral administration revealed a significant elevation of the fibrinogen degradation products (FDP) and euglobulin fibrinolysis, and an increase of t-PA antigen in the plasma, indicating release of endogenous plasminogen activator was probably from endothelial cell. The present findings suggest that NK may represent a useful and safe drug for fibrinolytic therapy by long-term intaking nattoorally.In this study, the nattokinase gene was cloned from the Bacillus natto genomic DNA by PCR as a 1146 bp fragment, encoding the signal peptide, precursor peptide and mature peptide.The cloned NK gene has a 100% homologue with the Bacillus subtilis var. natto subtilisin NAT (aprN) gene (genbank access No: S51909).The nattokinase gene was cloned into a prokaryon expression vector pET28a to construct a recombinant plasmid pETNK. The recipient cell of Escherichia coli BL21 was transformed with pETNK. NK was expressed under the induction of IPTG and identified by SDS-PAGE and Western blotting. The molecular mass of expressed rNK is about 31kD. An emulsion was prepared with the purified rNK and injected into rabbits. The polyclonal antibody in the rabbits serum is identified by ELISA and Western blotting. The fibrin plate assay indicated that the rNK fusion protein could cleavage fibrin effectively.On the other hand, the NK gene was inserted in the silkworm baculovirus expression vector system transfer vector pBacPAK8 and constructed the recombinant transfer vector pBacPAKNK. The purified pBacPAKNK DNA was co-transfected with linear virus Bm-BacPAK6 DNA into BmN cells. The homologous recombination occurred inside the cells then the recombinant virus...
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