Identification and characterization of a cathepsin-L-like peptidase in Eimeria tenella
Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in birds.Cysteine protease is a major virulence factor in parasitic protozoa, and it may be a suitable chemotherapeutic target and vaccine candidate molecule.A 100 amino acid (an.) partial sequence of cathepsin L, which is a cysteine protease, was reported by Katrib et al.(Ac.No.CDJ41293) (2012).A 219 an.sequence was reported by Reid et al.(Ac.No.AFV92863) (2013).However, the open reading frame (ORF) was not reported In this study, a full sequence of a cathepsin-L-like peptidase in Eimeria tenella (EtcatL) was obtained and its biochemical characterizations and expression profiles were analyzed across different stages of the parasite”s life cycle.Results showed that the EtcatL gene encodes a protein 470 aa.in length, with 47 and 49 % identity to Toxoplasma gondii and Eimeria acervulina.Considering the close phylogenetic relationship, TgcatL (PDB.ID 3F75) was selected for use as a template for homology modeling with quality factors of 90.9.Gelatin SDS-PAGE showed it to exert protease activity at ≈38 and ≈26 kDa.Further analysis showed the kinetic parameters of the recombinant peptidase to be Km-8.9 μM and Vmax--5.7 RFU/s μM at pH 5.5 containing 10 mM dithiothreitol (DTT) in the reaction matrix, and the IC50 value of E64 was 65.32±3.02 nM.The recombinant protein was active from 25 to 50 ℃, with optimal activity at 42 ℃.The RT-PCR and Western blot results showed it to be expressed mainly at the endogenous stages and the initial phase of the sporulation.The protective experiment showed that chickens immunized with 100 and 200 μg rEtcatL had reduction of weight loss values 48.7 and 57.9 % those of infected controls, respectively.Their reduction of lesion scores (RLS) were 25.0 and 47.2 % that of control chickens, and relative oocyst production (ROP) was 39.6 and 15.5 % that of control chickens.These results indicate that the EtcatL can be used as an effective immunogen,and further studies are needed to enhance its potential as a vaccine candidate molecule.
Eimeria tenella Cysteine protease Cathepsin L Enzyme kinetics Expression profile
Renqiang Liu Xueting Ma Aijun Liu Lei Zhang Jianping Cai Ming Wang
National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institude, Chine Key Laboratory of Veterinary Bioproduction and Veterinary Medicine of the Ministry of Agriculture, Z National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University,
国内会议
苏州
英文
201-214
2014-10-26（万方平台首次上网日期，不代表论文的发表时间）