| Paecitomyces lilacinus E7 is a strain of Nematophagous fungus isolated from soil and produced protease. The fermentable fluids of E7 strain can kill the nematode obviously.The fermentation medium and the conditions of a high yield alkaline protease were studied. The main components of the medium producing were substituted. The optimum medium was consist of: 2% bean powder,2% wheat bran, 1% NH4NO3, 0.5% peptone, 0.01% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, pH7.0. The optimum fermentation conditions were: inoculum volume 1%, a 150ml flask containing 30ml medium, 180r/min, 28℃.And the highest enzyme activity rose up to 465u/ml。The paper reviews enzymatic characteristics of protease about fermentation fluids.The optimum temperature and pH for protease acitivity were 40℃and pH10.5. It was stable in the pH range 8.0~11.0 and weak thermal stability.The primers was designed according to the conserved protease sequence of Paecitomyces lilacinus and other fungus. Sequence analysis showed that the homology between the cloned gene (500bp) of E7 and the reported PL gene was very high (100%). Based on the analysis, another specific primers were designed according to mature peptide sequence of the serine protease (PL) gene from Paecitomyces lilacinus. With the specific primers, the 850bp fragment was gained by RT-PCR.After digesting the fragment with BamH I/HindⅢ,PL gene was cloned into the expression plasmid vector pET-22b(+) digested with BamH I/HindⅢ.The recombinant vector (pET-22b–PL) was transformed into the expressing strain BL21(DE3) and induced expression by 1.mM IPTG at 37℃. The expected protein was 31kD and 60% for total protein.After SDS-PAGE,the recovery of PL added the same volume of incomplete Freund Adjuvant emulsified completely,injected rabbits by subcautaneous injection.After fourth injection ,blood was harvested by carotid artery and then antiserum was made from it.ELISA proved that the titres of the antiserum was 1/10000.
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