Cloning, Expression And Identification Of Cysteine Protease TsCP1of Trichinella Sprialis

The open reading frame (ORF) cDNA sequence of cysteine protease of T. spiralis (TsCP1) wasobtained by RT-PCR from total RNA of muscle larvae with primers derived from GenBank database T.spiralis EST sequences. TsCP1gene was cloned and analyzed with bioinformatics software. The resultsindicated that the TsCP1cDNA sequence contained an ORF of1101nucleotides and the deducedprotein consisted of366amino acids with the theoretical molecular weight of41.9ku and isoelectricpoint of7.46. Analysis of secondary structure revealed that α-helix and β-strands were31.4%and15.3%respectively. The signal peptide sequence of TsCP1located between amino acids1and19and3N-glycosylation sites were identified. The cysteine protease conserved active site of Cys173, His309and Asn333were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence wasrevealed in the propeptide of TsCP1, indicating TsCP1belongs to the cathepsin F subgroup of cysteineprotease family. Homology analysis revealed a higher than40%identity with other cathepsin F fromparasitic helminth and phylogenetic analysis indicated TsCP1located in the different clan withtrematode cathepsin Fs. RT-PCR revealed this gene is expressed in muscle larvae, newborn larvae andadult stages. TsCP1gene fragment was cloned into the prokaryotes expression vector pET30a and therecombinant pET30a-TsCP1was transformed into E.coli BL21(DE3) to induce expression.SDS-PAGErevealed the recombinant protein was expressed as inclusion body with the molecular weight of is45ku.The purified recombinant TsCP1was used to immunize rabbit and the immune serum could recognize a46ku band in soluble protein of muscle larvae. After renaturation the TsCP1recombinat proteindemonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH5.5and thisactivity could be inhibited by cysteine protease inhibitor E-64.All the above lays the foundation for thefurther study of the function of T. spiralis cysteine protease.