论文部分内容阅读
Background: Trichinellosis is a re-emerging infectious disease,caused by Trichinella spp.Cathepsin F belongs to cysteine protease that is a major virulence factor for parasitic helminths,and it may be a potential anti helminth drug target and vaccine candidate.The aim of this study was to done,express and identify a cathepsin F-like protease in Trichinella spiralis and to investigate its biochemical characteristics.Methods: The full-length cDNA encoding a putative cathepsin F-like protease in T.spiralis,TsCF1,was cloned and its biochemical characterization and expression profile were analyzed.Transcription of TsCF1 at different developmental stages of T.spiralis was observed by RT PCR.The recombinant TsCF1 protein was expressed by prokaryotic expression system and recombinant TsCF1(rTsCF1)was analyzed by western blotting.And expression of TsCF1 at muscle larvae stage was performed by immunofluorescent technique.Molecular modeling of TsCF1 and its binding mode with E-64 and K11777 were analyzed.Enzyme activity and inhibitory test with E-64 as inhibitor were investigated by using Z-Phe-Arg-AMC as specific substrate.Results: Sequence analysis revealed that TsCF1 ORF encodes a protein of 366 aa with a theoretical molecular weight of 41.9 kDa and an isoelectric point of 7.46.The cysteine protease conserved active site of Cys173,His 309 and Asn333 were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence was revealed in the propeptide of TsCF1.Sequence alignment analysis revealed a higher than 40%identity with other cathepsin F from parasitic helminth and phylogenetic analysis indicated TsCF1 located at the junction of nematode and trematode.RT-PCR revealed the gene was expressed in muscle larvae,newborn larvae and adult stages.SDS-PAGE revealed the recombinant protein was expressed with the molecular weight of 45 kDa.The purified rTsCF1 was used to immunize rabbit and the immune serum could recognize a band of about 46 kDa in soluble protein of adult,muscle larvae and ES product of muscle larvae.Immunolocalization analysis showed that TsCF1 located on the cuticle and stichosome of the muscle larvae.After renaturation rTsCF1 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64.Further analysis showed the kinetic parameters of rTsCF1 to be Km = 0.5091 μM and Vmax = 6.12 RFU/s μM at pH 5.5,and the IC50 value of E64 was 135.50± 16.90 nM.Conclusion: TsCF1 was expressed in all stages of T.spiralis and localized in the cuticle and stichosome.TsCF1 might play a role in the life cycle of T.spiralis and could be used as a potential vaccine candidate and drug target against T.spiralis infection.