Comparison Of RCystatin Characteristics Of Haemaphysalis Flava

Cystatin(Cystatin)is an inhibitor that acts on cysteine protease and loses its enzymatic activity.It participates in important physiology such as blood meal,innate immunity,and immune regulation of the host.Studies have shown that Cystatin plays an important role in the invasion of parasites.In addition,the chemical Cystatin has the effect of inhibiting the growth of Pyricularia and helping the treatment of Plasmodium.Cystatin has four subfamilies,and its family 2 is widely found in the salivary glands,ovaries and other organs of ticks.It is speculated that it is involved in physiological processes such as full blood,blood digestion,and embryonic development.In this study,we will study some the Cystatin family 2 of Haemaphysalis Flava and explore the inhibitory effect of prokaryotic expression of rCystatin on cysteine proteases.1.Amplify the 5 ’and 3’ ends of Contig5433 by RACE method to obtain its full length,named Hfcyst-5;full length 700 bp,ORF frame length 399 bp,encoding 133 aa,1-17 aa as signal peptide,structural analysis showed for the Cystatin family 2.2.Insert Hfcyst-2,Hfcyst-5 into pET32 a,Hfcyst-2,Hfcyst-3,Hfcyst-5 into p GEX-4T-1,pick the positive plasmid and transform into BL21(DE3),and successfully express the recombinant protein induced r Hfcyst-2 、 r Hfcyst-3 and r Hfcyst-5 by IPTG.SDS-PAGE detection revealed that the main expression form of recombinant proteins expressed by pET32 a or p GEX-4T-1 was inclusion bodies.3.Denaturation of the expressed inclusion bodies of r Hfcyst-2,r Hfcyst-3,and r Hfcyst-5 revealed that r Hfcyst-5 expressed by p GEX-4T-1 was insoluble in 8M urea;purified and renatured to obtain pET32 a expressed r Hfcyst-2 and r Hfcyst-5 Protein solution,purified r Hfcyst-2 and r Hfcyst-3 protein expressed by p GEX-4T-1 And the inhibitory effect of r Gystatin expressed by p GEX-4T-1 was slightly lower than that of rCystatin expressed by pET32 a.