Identification and characterization of a cathepsin-L-like peptidase in Eimeria tenella

文献类型： 外文期刊

第一作者： Liu, Renqiang

作者： Liu, Renqiang;Liu, Aijun;Wang, Ming;Ma, Xueting;Cai, Jianping;Zhang, Lei;Wang, Ming

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关键词： Eimeria tenella;Cysteine protease;Cathepsin L;Enzyme kinetics;Expression profile

期刊名称：PARASITOLOGY RESEARCH （ 影响因子：2.289； 五年影响因子：2.403 ）

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收录情况： SCI

摘要： 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 (aa.) partial sequence of cathepsin L, which is a cysteine protease, was reported by Katrib et al. (Ac. No. CDJ41293) (2012). A 219 aa. 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 proteaseactivity at ≈38 and ≈26 kDa. Further analysis showed the kinetic parameters of the recombinant peptidase to be K_m=8.9 muM and F_(max)=5.7 RFU/s muM at pH 5.5 containing 10 mM dithiothreitol (DTT) in the reaction matrix, and the IC_(50) value of E64 was 65.32±3.02 nM. The recombinant protein was active from 25 to 50 °C, with optimal activity at 42 °C. 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 mug 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.

分类号： R53

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