Purification Of Nattokinase And Preparation Of Monoclonal Antibody Against Nattokinase From Natto

Nattokinase which had fibrinolytic activity, produced by Bacillus subtilis natto during fermentation, is a kind of serine enzyme. Studies showed that nattokinase could obviously dissolve fibrin, blood clot, enhance fibrinolytic system's function of animal and human thrombus model. Nattokinase from fermented food with high efficiency of fibrinolytic was stable in enteron and absorbable by intestine. So development of nattokinase for healthy food and oral thrombolysis medicament had a bright prospect. Preparation of monoclonal antibody against nattokinase should be useful in quantitative analysis and identification of nattokinase.Crude nattokinase was purified through procedures including ammonium sulfate differential precipitation, dialysis, inspissation, sephadex G-100, CM-Sepharose FF chromatography. After purification, the total protein was reduced from 106723 UK unit to 5112 UK unit, but the specific activity was rised from 21.43 UK u/mg protein to 709.2 Uk u/mg protein and was purified by 33.09 times. The purified nattokinase showed single protein band on gels of SDS-PAGE.The BALB/c mice were immunized four times with the purified nattokinase, then the spleen cells of the hyperimmunized mouse and SP2/0 cells were fused with PEG-1500. After cell fusion, 98 positive holes were obtained. Two hybridoma cell monoclonal antibodies, named NA3, NT5 were developed by twice subclones. NA3 belonged class IgG, subclass IgG2a. The titers of its culture and ascite were 1: 10~6 , identified by indirect enzyme-linked immunosorbent assay (ELISA). After four month repeated frozen storage, thawed revival and generation, NA3 and NT5 were steadily producing high titer of Mabs to nattokinase.In indirect ELISA, two monoclonal antibodies were specific for Bacillus natto.It is believed that the monoclonal antibodies against nattokinase should be helpful in developing the medicine of nattokinase.