Molecular Cloning And Prokarytic Expression Of Nattokinase Gene

Recently brain-cardiovascular thrombus disease become more and more harmful to people. Thrombosis has been becoming the second-deadly disease next to carcinoma and its incidence grew up steadily. What makes it more serous is that patients who catch the disease become younger and younger. So it's gets more urgent to find innoxious drugs which can be used not only in the treatment of embolism but also in the prevention of the disease.Nattokinase (NK) is a new fibrinolytic enzyme that has been intensively studied in recent years, which directly cross-linked fibrin in vitro. It was firstly founded in a traditional Japanese fermented food called"natto". In vitro and in viro, NK has been observed to thrombus soluble by decompose interstrand cross-link fibrous protein.The thrombolytic activity of NK is 4 times that of plasmin and cannot cause hemorrhage. Now researchers all over the world has begun to do some work on DNA sequence of NK and express system of protein enzymes which have high homology with NK.In this dissertation, associated bacteria with plasmin made by ourselves previously was isolated by using the fibrin plate method. After purification, the most activated bacillus which had a largger transparent loop around the fungi was chosen to product fibrinolytic enzyme plasmin and was numbered KX+1 after identification.A primer based on NK cDNA was designed. NK gene were amplification by PCR, using this primer and subtillus total DNA。pBV-NK was constructed by link plasmid pBV220 and NK gene.Both restriction digesting pBV-NK with EcoR1 and BamH1,one major band of 757bp was observed on agarose electrophoresis. further confirmation by DNA sequencing of PCR product and by comparing sequencing results with DNA sequence of NK showed that pBV-NK was NK gene. The nucleotide homology was 99.88%.The pBV-NK plasmid was transformed into E.coli. SDS-PAGE analyzing showed Nk cDNA was expressed in E.coli and its molecular weight was 28000Da.NK gene was cloned and its expression plasmid was constructed by using molecular clone and gene reconstruction technology. A NK engineering strain which had activity og decompose thrombus was obtained. This work can be further used in producing NK with genetic hecombinant strain.