Cloning, Expression And Functions Of Babesia Orientalis Heat Shock Protein20

Babesia orientalis is a hemoparasite of buffalo which is transmitted by Rhipicephalus haemaphysaloides. The clinical symptoms caused by B. orientalis is characterized by fever, anemia, jaundice, hemoglobinuria and even mortality. Buffalo babesiosis caused by B.orientalis is one of the most severe diseases of buffalo in central and south China. The studies in our lab on the morphology, life cycle, vector and molecular taxonomy of B.orientalis have verified that B.orientalis is a new species of Babeisa family. Recently, we are focused on the molecular biology characteristics of B.orientalis thus we have sequenced the genome of B.orientalis, which will facilitate the researching of functional genes of B.orientalis.The family of small heat shock proteins (sHSPs) comprises the most diverse group of HSPs, the molecular weight of which varies from12to43kDa. The family is well characterized by a strictly conserved a-crystallin domain. Parasite sHSPs have great impact on the infection process, for example, HSP20gene is recently shown to play important roles in migration and invasion of apicomplexan parasites. The well conserved babesial HSP20is antigenic and could be recognized by memory CD4+T lymphocytes from babesia-immune cattle. In the present study, we obtained a HSP20homologous in the complete B. orientalis genomic database. We cloned this gene and the sequence was analyzed. Then the prokaryotic expression of HSP20gene was carried out. The reactogenicity and chaperone activity of the HSP20protein were determined. The works are described as follows.(1) Cloning and sequence analysis of B.orientalis HSP20Specific primers were designed based on the HSP20fragment obtained after searching the B. orientalis genomic database. A690bp full length HSP20gene was amplified from B.orientalis genomic DNA, which was named as BoHsp20. The BoHsp20contains a single intron with an ORF of534bp which encoding177amino acid residues. A a-crystallin domain was presentd in the deduced protein sequence, hence the BoHSP20protein belonged to the sHSPs family. Homology analysis showed that BoHSP20sequence was most similar to the HSP20protein sequence of B. bovis, B.bigemina and B.divergens, with the identity of94%,86%and86%, respectively. Phylogenetic analysis showed that HSP20sequence identified in the present study fell into a sister group with B. bovis which indicating the genetic relation between these two species was most closely. Phylogenetic analysis also showed that B. orientalis was a new species. Bioinformatics analysis revealed great antigenicity of BoHSP20.(2) Prokaryotic expression, purification and antigenicity of B.orientalis HSP20The recombinant plasmid pET-28a-BoHSP20was successfully constructed and expressed in E.coli BL21. SDS-PAGE analysis revealed an approximately20kDa band as expect. The his-tag fused BoHSP20protein was purified with affinity chromatography column. Western blot analysis showed that the purified protein could specifically react with serum from B.orientalis-infected buffalo, which indicate the antigenicity of rBoHSP20.(3) The reactogenicity of B.orientalis HSP20Indirect ELISA based on rBoHSP20was done to detect the antibody level of serum from B.orientalis experimentally infected buffalo. Results showed the B.orientalis antibody could be detected16days post infection. The antibody level retained high until62days post infection. These results indicated that BoHSP20elicited humoral immunity during B.orientalis infection.Rabbit polyclonal antibodies against BoHSP20were prepared and were applied to indirect immunofluorescent assay. The polyclonal antibodies recognized HSP20on the surface of merozoites in the red blood cells. These indicated that HSP20could be used in indirect immunofluorescence assay of B.orientalis.All the results showed that B.orientalis HSP20was reactogenic.(4) Chaperone activity of B.orientalis HSP20The viability of E.coli cells expressing BoHSP20protein were increased more than twofold when exposed to50℃thermal stress, which means that overexpression of BoHSP20protein confers a significant degree of thermotolerance to E.coli. In vitro, BoHSP20prevent the aggregation of BSA caused by chemical denaturant. These indicated that BoHSP20performs chaperone activity.In the present study, B. orientalis HSP20gene was successfully cloned, sequence analysis revealed the gene was conserved among the babesial parasite. Western blot, ELISA and IFA analysis showed that BoHSP20was reactogenic. The chaperone activity also proved that HSP20was a functional gene of B. orientalis, which providing a theoretical basis for diagnosing and preventing of babesiosis.