Sequence Analysis Of TRAP And HSP70 Genes Of Babesia Gibsoni In Dogs And The Study On Antigenicity Of Recombinant HSP70 Protein

Babesiosis is a tick-borne disease,which transmitted through aphids.Clinical manifestations are characterized by fever,anemia,hemoglobinuria as well as death.Babesia gibsini is one of the most important pathogen caused piroplasmosis in China.In 1985,the first case of Babesia was discovered and confirmed in China.In recent years,there have been more and more cases of Babesia infection in companion dogs and police dogs.The situation in various veterinary hospitals can also confirm that the disease in our country is widely popular.At present,there is no vaccine to prevent Babesia,and the method is relatively simple in the diagnosis and treatment of the disease.Heat Shock Proteins(HSPs)is a type of heat stress protein in nature.The protein is highly conserved and can induce antibodies in the body.The study suggests that HSP70 protein can be used as a diagnostic tool for Babesia detection and as a vaccine candidate protein.Therefore,make research on HSP70 gene and its encoded protein has become a hotspot in the diagnosis and prevention of Babesia.The research work includes the following two parts:1.Sequence analysis of the TRAP gene and HSP70 gene of Babesia gibsoni isolated from dogs in Nanjing,ChinaIn this study,the TRAP gene and the HSP70 gene of Babesia gibsoni isolated from the naturally infected dog in the Nanjing area were cloned and sequenced,and compared with other isolates in Asian.The similarity of TRAP and HSP70 gene sequences among four isolates in Nanjing were above 99%.The Nanjing isolates were closely related to isolates from Taiwan and Japan.Indian isolates were different form Chinese and Japanese isolates.These results suggest that the TRAP and HSP70 genes have a reference value for the genetic diversity analysis of Babesia gibsoni.2.Prokaryotic expression and antigenic characteristics of HSP70 Gene of Babesia gibsoniHeat shock protein 70(Heat shock protein,HSP70)gene of Nanjing isolate was cloned into pET-32a plasmid.The recombinant plasmid was transformed into BL21(DE3),which was then induced by IPTG to produce the HSP70 protein of Babesia gibsoni.The HSP70 protein was purified by Ni column and was used to immunize 6 week old BalB/C mice to prepare antiserum.The results of Western blot showed that the recombinant protein had excellent reactivity and immunogenicity.The recombinant protein not only reacted specifically with the sera of dogs naturally infected by Babesia gibsoni,but also stimulated the production of antibodies with high titers in mice.