Cloning,Expression And Anti-inflammation Activity Of Thermophilic Serine Protease Inhibitor-pnserpin

Serine protease inhibitors(serpins)are native inhibitors of serine proteases,constituting a large protein family with members spread over animals,plants and microorganisms.They are natural inhibitors of serine proteases,involved in a variety of important life processes,such as coagulation,complement activation,inflammatory response,cell migration,cell matrix reconstruction and tumor suppression.At present,the serpins dysfunctional diseases are mainly treated by complementary therapies.The clinical application of serpins is mainly obtained by human plasma purification,which cost is very high,and is not conducive to the wide application.The diversity of microorganisms provides an inexhaustible motive force for the discovery of new drugs.Proteins derived from the thermophilic microorganisms and their origins are highly stable,and can be developed for more valuable bioactive substances.In this paper,the properties and bioactivity of the serine protease inhibitor from Pyrobaculum neutrophilum(Pnserpin)were studyed,to provide a new candidate drug molecule for the treatment of serpins and inflammation-related diseases.1.Cloning,expression,purification and characterization of Pnserpin: The gene of Pnserpin was cloned into pET-28 a expression vector and then transformed into Escherichia coli BL21 to induce the expression of the protein.The optimal expression conditions of Pnserpin were explored,and the protein was purified by nickel affinity column chromatography.Using serine protease and cysteine protease as substrate,the basic kinetic constants and the optimum reaction temperature,pH and thermal stability of Pnserpin were determined.Our results indicate that Pnserpin is a highly thermostable serine protease inhibitor.2.The anti-inflammatory activity of Pnserpin: The anti-inflammatory effect of different concentrations of Pnserpin in vivo was studyed using xylene-induced mouse ear swelling.In order to study the anti-inflammatory activity of Pnserpin at the cellular level,we investigate the effect of Pnserpin on the expression of proinflammatory cytokines such as TNF-?,IL-1? and IL-6,using lipopolysaccharide(Lipopolysaccharide,LPS)stimulated mouse macrophage cell RAW264.7.The results showed that Pnserpin had a significant inhibitory effect on xylene-induced mouse ear swelling,indicating that Pnserpin had anti-inflammatory activity in vivo;The results at cellular level showed that Pnserpin significantly inhibited the expression of TNF-?,IL-1? and IL-6 in LPS-induced RAW264.7 cells.These experimental results indicated that Pnserpin had anti-inflammatory activity at the cellular and animal levels and could be further developed and applied.