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Molecular characterization and analysis of a novel calcium-dependent protein kinase from Eimeria tenella

Published online by Cambridge University Press:  01 February 2013

H.Y. HAN
Affiliation:
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, China
S.H. ZHU
Affiliation:
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, China
L.L. JIANG
Affiliation:
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, China
Y. LI
Affiliation:
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, China
H. DONG
Affiliation:
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, China
Q.P. ZHAO
Affiliation:
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, China
C.L. KONG
Affiliation:
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, China
B. HUANG*
Affiliation:
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, China
*
*Corresponding author. Tel: +86(0)21 34293464. Fax: +86(0)21 54081818. E-mail address: hb@shvri.ac.cn

Summary

The calcium-dependent protein kinases (CDPKs) are unique enzymes found only in plants, green algae, ciliates and apicomplexan parasites. In this study, a novel CDPK gene of Eimeria tenella, designed EtCDPK3, was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). The entire cDNA of EtCDPK3 contained 1637 nucleotides encoding 433 amino acids and the deduced EtCDPK3 protein had canonical characteristic domains identified in other CDPKs, including a well-conserved amino-terminal kinase domain and a carboxy-terminal calmodulin-like structure with 4 EF-hand motifs for calcium binding. The expression profiles of the EtCDPK3 gene in different development stages were investigated by real-time quantitative PCR. Messenger RNA levels from the EtCDPK3 gene were higher in sporozoites than in other stages (unsporulated oocysts, sporulated oocysts and merozoites). Western blot analysis showed that rabbit antiserum against recombinant EtCDPK3 could recognize a native 49 kDa protein band of parasite. Indirect immunofluorescent antibody labelling revealed dispersed localization of EtCDPK3 during the first schizogony and intense specific staining. EtCDPK3 was located at the apical end of the sporozoites after early infection of DF-1 cells and the protein was highly expressed. Inhibition of EtCDPK3 function using specific antibodies reduced the ability of E. tenella to invade host cells. These results suggested that EtCDPK3 may be involved in invasion and survival of the parasite intracellular stages of E. tenella. Because this kinase family is absent from hosts, it represents a valid target that could be exploited for chemotherapy against Eimeria spp.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2013

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