Cloning, Expression Of Babesia RAP-1C And The Establishment Of An Indirect ELISA Of B.bigemina

Babesiosis is the general term of protozoosis in vertebrate, which is caused byparasites of Babesia genus parasiting in red cells of hosts. The disease brings seriousharm to the livestock industry and human’s health with the main clinical features suchas fever, oligocytosis, anemia, jaundice, hemoglobinuria, visual mucosal bleeding, etal, and the incidence of serious can cause animal death some times. Bovie Babesiosisis major caused by B.bigemina and B.bovis parasitizing in cattle. B.bovis andB.bigemina cause the highest incidence and mortality, and both of them share thesame vector tick-Rhipicephalus microplus in China, and lead to mixed infection,which is difficult to distinguish in clinic. Therefore, it is of great significance to carryout the differential diagnosis of the mixed infection.In this study RAP-1C fragment of B.bovis and B.bigemina were prokaryoticexpressed and reactogenicity was developed using the recombinant proteins. Themajor works were presented as following:1. The analysis, cloning and expression of RAP-1C gene of B.bigemina andB.bovisThe coding sequence and antigen epitope of rhoptry-associated protein (RAP-1)of B.bigemina&B.bovis were analyzed and find that there are low similarity and highantigen epitope region of C-terminal fragment.The RAP-1C genes of B. bigemina（No.313-484）and B.bovis（No.317-537）were cloned and amplified with specificprimers, and they were prokaryotic expressed by using recombinant plasmid. Theexpressed products were confirmed to be about26ku and31.9ku soluble bySDS-PAGE.2. The purification and reactogenicity analysis of the recombinant proteins ofRAP-1C of B.bigemina and B.bovisThen the recombinant proteins were purificated by Ni-NTA purification system,and the concentration of B.bigemina&B.bovis were0.4mg/mL and0.8mg/mL.Thereactionogenicity and specificity of RAP-1C of B.bigemina and B.bovis wereanalyzed by Western-blot. Their reactionogenicity were identified and there was notcross-reactivity between recombinant proteins and positive sera of B.major、T.segentiand T.annulata.4. The establishment of an indirect ELISA of B.bigeminaAn indirect ELISA specific for B.bigemina was established with the purifiedrecombinant protein. The result showed that the specificity and sensitivity of thismethod were95.5%and100%respectively, when0.6363of OD450was chosen aspositive threshold via analyzing the data of20negative sera and90positive sera with MedCalc software. And there is no cross-rection between RAP-1C of B.bigemina andthe other major seras of piroplasmida of cattle.