title: Molecular cloning and characterization of Babesia orientalis rhoptry-associated protein 1.
abstract: The rhoptry-associated protein 1 (RAP-1) gene of Babesia orientalis was obtained from a cDNA expression library by immunoscreening with B. orientalis-infected water buffalo sera. The nucleotide sequence of the cDNA was 1732 bp with an open reading frame (ORF) of 1434 bp, encoding a polypeptide of 478 amino acid residues with a predicted size of 52.5 kDa. The ORF was cloned into a pGEX-KG plasmid and subsequently expressed as a GST-fusion protein. The recombinant RAP-1 of B. orientalis (rBoRAP-1) was purified and evaluated as an antigen using Western blotting. The native BoRAP-1 was recognized by the antibodies raised in rabbits against rBoRAP-1. Strong immunofluorescence signals were observed in erythrocytes infected with B. orientalis. Phylogentic analysis revealed that B. orientalis fell into a Babesia clade and most closely related to Babesia bovis and Babesia ovis, which was similar to the previous reported trees based on 18S rRNA and HSP70 genes. The present study suggests that the BoRAP-1 might be a potential diagnostic antigen, and the RAP-1 genes can aid in the classification of Babesia and Theileria species.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China. Electronic address: helan@mail.hzau.edu.cn.
authors.forename: Lan
authors.initials: L
authors.lastname: He
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Wen-Jie
authors.initials: WJ
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Jian-Xi
authors.initials: JX
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Jin-Fang
authors.initials: JF
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Miao
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Li-Zhe
authors.initials: LZ
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Muhammad Kasib
authors.initials: MK
authors.lastname: Khan
authors.affiliation: Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Yan-Qin
authors.initials: YQ
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
authors.forename: Min
authors.initials: M
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China; Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China. Electronic address: zhaojunlong@mail.hzau.edu.cn.
authors.forename: Jun-Long
authors.initials: JL
authors.lastname: Zhao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology*; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001404:Babesiosis / Q000175:diagnosis* / Q000469:parasitology; D001483:Base Sequence; D002020:Buffaloes / Q000469:parasitology*; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D015723:Gene Library; D008969:Molecular Sequence Data; D010802:Phylogeny; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D017422:Sequence Analysis, DNA / Q000662:veterinary
chemical_list: D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D015800:Protozoan Proteins; C097551:rhoptry-associated protein-1, Babesia
keywords: Antigenicity; Babesia orientalis; Diagnosis; Rhoptry-associated protein