Cloning And Recombinant Expression Of Three Immuno-related Genes Of Babesia Orientalis

Babesia orientalis, as a tick-borne hemoprotozoan parasite, is transmitted by Rhipicephalus haemaphysaloides. It is a causative agent of buffalo babesiosis and is found to the south of Yangtse River. The clinic manifestations are fever, anemia, icterus, hemoglobinuria and frequent mortality. Though a thorough understanding has been made about its culture in vitro, morphology, transmission, molecular taxonomic and diagnosis study, there are still blanks in the invasion mechanism of the pathogen and effective candidates to prevent the disease.In the present study,18positive clones were obtained by immunscreening B. orientalis cDNA expression library. Three of the immuno-related genes were cloned, expressed and molecular characterizations were analyzed.（1） Immunological screen of B. orientalis cDNA libraryA cDNA expression library constructed from B. orientalis merozoites was immunoscreened by the anti-B. orientalis buffalo serum, and three immuno-related clones （BoP34, BoP21and AMA1） were obtained.The complete nucleotide sequence of BoP34cDNA was1088bp with two open reading frames （ORFs）. The introless BoP34gene encoded251amino acid residues with a predicted molecular weight of34kDa. BLAST analysis showed that the nucleotide sequence of BoP34had72%similarity with that of Babesia bovis nuclear movement family protein （XM₀01611335）. The results suggested that BoP34could be a member of the nuclear movement family.The complete nucleotide sequence of BoP21cDNA was732bp with a579bp ORE The introless BoP21gene encoded192amino acid residues with a predicted molecular weight of21kDa. BLAST analysis showed that the nucleotide sequence of BoP21had68%similarity with that of B. bovis hypothetical protein （BAN64167.1）. The results suggested that BoP21could be a member of the hypothetical family.The complete nucleotide sequence of AMA1cDNA was1803bp encoding600amino acids, and the predicted molecular weight of AMA1was67kDa. Homology analysis showed that Bo AM A1sequence was most similar to that of B. bovis （GenBank accession number:XP001611043）, B. bigemina （GenBank accession number:ADP02977） and B. gibsoni （GenBank accession number:ABD04040）, with the identify of72%,52%and53%. Structure analysis showed that the predicted amino acid sequence had characteristics belonging to Babesia AMA1family, such as a signal peptide at N-terminal, cysteine residues that formed disulfide bonds, an extracellular domain including three structural domains （I/II/III）, a transmembrane region at C-terminal and a cytoplasmic region.（2） Cloning and prokaryotic expression of BoP34, BoP21and AMA1According to the sequencing results of full-length BoP34, BoP21and AMA1, specific primers were designed to clone BoP34-ORF2, BoP21-ORF and domain Ⅰ/Ⅱ/Ⅲ of AMA1. The amplicons were cloned into prokaryotic expression vectors. Recombinant plasmids pET-32a-BoP34-ORF2, pET-28a-BoP21-ORF and pET-28a-AMA1-DI/II/III were constructed and highly expressed in E. coli BL21. The purified recombinant proteins of BoP34, BoP21and AMA1were used to produce polyclonal antibodies of anti-BoP34, BoP21or AMA1in Japanese white rabbits. ELISA showed that the titer of these antibodies were100×27,100×27and100×28, respectively. Western blot showed that the anti-B. orientalis buffalo serum was able to react with rBoP34, rBoP21or rAMA1, indicating that each protein was an immunodominant antigen. Antibodies of anti-BoP34, BoP21or AMA1were also immunobloted with total proteins of B. orientalis merozoites to detect BoP34, BoP21and AMA1in vivo, respectively. The experimental results showed that the native BoP34, BoP21and AMA1were expressed in B. orientalis merozoite stage.（3） Eukaryotic expression of truncated B. orientalis AMA1According to the location of disulfide bonds, the extracellular domain of B. orientalis AMA1was divided into three structural domains （Ⅰ/Ⅱ/Ⅲ）. Domain Ⅰ, Ⅱ, Ⅲ, Ⅰ/Ⅱ, II/III and I/II/III with flag tags were recombined into eukaryotic expression plasmid pT8. These plasmids were transfected into COS-1cells by liposomes for expression. After transfection of48hours, all the plasmids were expressed successfully on the cell membrane detected by indirect immunofluorescence assay.In summary, three immuno-related genes-BoP34, BoP21and AMA1, were obtained by immunoscreening the B. orientalis cDNA expression library. They were cloned, expressed, and the corresponding polyclonal antibodies were produced, respectively. Good antigenicity and high quantity of native proteins in B. orientalis merozoites were also demonstrated in our study. All the results supported that BoP34, BoP21and AMA1could be candidate antigens for detection of B. orientalis antibodies.