Cloning, Expression And Identification Of Cathepsin B Gene Family Of Trichinella Spiralis

By analysis of Trichinella spiralis genome and EST databases in GenBank, four membersof cathepsin B family (TsCB1～4) were obtained and specific primers were designed. Theopen reading frame (ORF) cDNA sequences of TsCB1～4were cloned from total RNA of T.spiralis muscle larvae by RT-PCR and bioinformatic analysis were performed. The resultsshowed TsCB1～4owned typic cysteine protease domain and conserved active sites exceptthat the active site cysteine changed into serine in TsCB1, indicating them belonged to thecathepsin B family of cysteine protease clan. RT-PCR results revealed TsCB1～4weretranscribed in newborn larvae, muscle larvae and adult of T. spiralis while the transcriptionallevel of TsCB1and TsCB2were lower in5-day-old adult and newborn larvae, respectively.The phylogenetic analysis indicated TsCB1～3were close to those from non-blood feedingparasitic nematodes while TsCB4located in the same clan with blood-feeding helminthes,which indicated the multiple functionsof cathepsin B during evolution. The gene fragmentsencoding proenzyme of TsCB1～4were cloned into pET-30a and pMAL-c2x respectively, andexpressed in E. coli BL21(DE3) after induced by IPTG and recombinant proteins were about57、43、47and80ku as insoluble inclusion bodies. TsCB4recombinant protein could berecognized by T. spiralis infected pig serum and showed reactivity. Antiserum againstrecombinant TsCBs could recognize the36,30,33,30bands in muscle larvae respectively.TsCB2and TsCB3recombinant proteins showed the catalytic activity to cleave gelatin afterrenaturation and the activity could be inhibited by specific cysteine protease inhibitor E-64.This will lay the foundation for the further study of functuions of catheptin B in T. spiralis.