The Studies On Biological Characterization Of Cathepsin B And L In Eimeria Tenella

Cathepsin B and cathepsin L cysteine proteases are main members of papain-like protease family, which are diversely widespread and have extensive biological functions. In parasitology, cathepsin B and cathepsin L have been reported in many apicomplexa protozoa, and take part in the invasion, evasion, transition, excystation, and nutrient uptake.However, there are few research reports inE. tenella.In this study, we successfully cloned the cDNA sequence of cathepsin B (Etcat B) and cathepsin L (Etcat L) in Eimeria tenellausing RT-PCR and RACE. The sequence of Etcat B is1539bp, and contains the complete open reading frame (ORF) coding512amino acids, shares49%identity with Toxoplasmagondii’ cathepsin B (Ac NO. EPR63374.1). Meanwhile, the sequence of Etcat L is2176bp, and includes the open reading frame of1413bp coding470amino acids, itshares49%identity with Eimeriaacervulina’ cathepsin L (Ac No. CDI79476) and47%identity with T. gondii (Ac. No. ABY58967.1). Using prokaryotic expression system, We cloned and purifiedthe recombinant Etcat B and Etcat L proteins, and confirmed enzyme activity of both proteins using SDS-PAGE enzymograph. Then, BALB/c mices were immunized with both proteins, and the antibody IgG titer was detected by ELISA. With the method of real-time PCR and Western blot, we detected the expression of Etcat B and Etcat L at different stage. The result suggested that Etcat B was higher in the phase of gametophyte and merozoite, and gradually increased during sporulation process. Etcat L mainly expressed in the merozoite and gametophyte phase, and increasingly expressed at the initial of the sporulation. The experiment laid a solid foundation for the role of Etcat B and Etcat L in parasite development and host pathogenic ity.Further analysis revealed the kinetic parameters of the recombinant Etcat L to be Km=8.9μM and Vmax=5.7RFU/s-μM in the reaction buffer containing10mM dithiothreitol (DTT), pH5.5and the IC50to E64was65.32±3.02nM. Temperature impact on Etcat L activity implied that recombinant proteins had activity between25℃to50℃, with optimal activity at42℃, which corresponded to parasitic intestinal environment.The protective experiment showed that chickens immunized with100μg and200μg rEtcatL had reduction of weight loss values by48.7%and57.9%compared with those of infected controls, respectively. Their lesion scores (RLS) were reduced by25.0%and47.2%compared with that of control chickens, and relative oocyst production (ROP) was at the range of39.6%and15.5%. 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.Cloning and expression the cathepsin B&L in E. tenella, provide the theoretical basis for further exploration of the role of the gene in chicken coccidia invasion and other biological processes.