Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.
The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
Qi
Q
Zhai
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, PR China.
Bing
B
Huang
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
Hui
H
Dong
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
Qiping
Q
Zhao
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
Shunhai
S
Zhu
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
Siting
S
Liang
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
Sha
S
Li
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
Sihan
S
Yang
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
Hongyu
H
Han
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002432:Cecum / Q000276:immunology / Q000469:parasitology / Q000648:ultrastructure; D002460:Cell Line; D002645:Chickens / Q000276:immunology* / Q000469:parasitology; D003001:Cloning, Molecular; D003048:Coccidiosis / Q000276:immunology / Q000469:parasitology / Q000517:prevention & control* / Q000662:veterinary; D017124:Conserved Sequence; D016786:Eimeria tenella / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000276:immunology / Q000469:parasitology / Q000648:ultrastructure; D015870:Gene Expression; D007106:Immune Sera / Q000737:chemistry / Q000302:isolation & purification; D007114:Immunization; D052940:Merozoites / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008970:Molecular Weight; D033661:Oocysts / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D016366:Open Reading Frames; D011201:Poultry Diseases / Q000235:genetics / Q000276:immunology / Q000469:parasitology / Q000517:prevention & control*; D015800:Protozoan Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D034101:Sporozoites / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
D007106:Immune Sera; D015800:Protozoan Proteins; D011994:Recombinant Proteins