title: Molecular cloning and characterization of a novel heat shock protein 20 of Babesia orientalis.
abstract: The heat shock protein 20 (HSP20) gene of Babesia orientalis (BoHSP20) was identified from both genomic DNA and cDNA. The full-length BoHSP20 gene was 690bp with one intron from position 88-243bp. The amplicon obtained from cDNA corresponded to a full-length open reading frame (ORF) with a length of 534bp, encoding a polypeptide of 178 amino acid residues with a predicted size of 20kDa. The ORF was cloned into a pET-28a plasmid and subsequently expressed as a His-fusion protein. The recombinant HSP20 of B. orientalis (rBoHSP20) was purified and evaluated as an antigen using Western blotting. Anti-B. orientalis water buffalo serum reacted with rBoHSP20, indicating that this protein was an immunodominant antigen and could be a useful diagnostic reagent to detect antibodies against B. orientalis in water buffalo. The native BoHSP20 was recognized by polyclonal antibody from the serum of rabbit immunized with rBoHSP20. Strong immunofluorescence signals were observed from B. orientalis in blood smears by fluorescence microscopy. Bacterial survival experiments indicated that HSP20 can significantly increase the viability of bacteria when the culture is exposed to thermal stress. The results suggest that BoHSP20 might play an important role during B. orientalis transmission from tick to host animal, given the sudden shifts in temperature involved. Phylogenetic analysis revealed that B. orientalis is in the Babesia clade and most closely related to Babesia bovis. Similar topologies were obtained from trees based on 18S rRNA and the HSP70 gene. The present study suggests that BoHSP20 might be a potential diagnostic antigen and that the HSP20 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, PR China.
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, PR 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, PR 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, PR China.
authors.forename: Qing-Li
authors.initials: QL
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, PR 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, PR 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, PR China.
authors.forename: Muhammad Kasib
authors.initials: MK
authors.lastname: Khan
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China; Key Laboratory of Animal Epidemical Disease and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, PR 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, PR China; Key Laboratory of Animal Epidemical Disease and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, PR 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, PR China; Key Laboratory of Animal Epidemical Disease and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China. Electronic address: zhaojunlong@mail.hzau.edu.cn.
authors.forename: Jun-Long
authors.initials: JL
authors.lastname: Zhao
mesh_terms: D000818:Animals; D001403:Babesia / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D001404:Babesiosis / Q000469:parasitology*; D002020:Buffaloes / Q000469:parasitology*; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis; D050886:HSP20 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D013047:Specific Pathogen-Free Organisms
chemical_list: D018076:DNA, Complementary; D016054:DNA, Protozoan; D050886:HSP20 Heat-Shock Proteins; D015800:Protozoan Proteins
keywords: Babesia orientalis; Babesiosis; Heat shock protein 20; Phylogenetic tree