Cloning And Eukaryotic Expression Of Gene Rap-1of Bovine Babesia Bigemina

Babesia bigemina are tick-borne apicomplexans that mμltiplies in red blood cells.The bovine babesia bigemina is reportedly the one of the severest parasitosis that does harm to the world cattle industry. Major clinical presentation includes the number of red cells decrease, anemia, jaundice, hemoglobinuria, Finally can cause a host death.The top complex organelles of Babesia bigeminacan directly cause entity invasion the host cell, for instance, rap-1protein, this studies show that rap-1gene is a potential target for drμg screening and laid a good foundation for screening model in Babesia bigemina.The following studies were conducted in the present article:1. rap-1gene cloning and sequence analysis:According to rap-1gene sequence of Babesia bigemina in Genbank, primers were designed and synthesized. The genomic DNA of Babesia bigemina was extracted. The rap-1gene was amplified by polymerase chain reaction (PCR), The amplified fragment was inserted into pGEM-T Easy Vector, the complex was sequenced and identified, The resμlts showed that the Babesia bigemina rap-1gene has846bp basic radical. The allogenic analysis indicated the homology between cloning sequence and reference sequence on the GenBank was as high as99.74%. Further showed that the rap-1gene was high conservative in Babesia bigemina.2. Eukaryotic expression of rap-1gene of Babesia bigeminaBabesia bigemina pGEM-T-rap-1plasmid from the laboratory, after the PCR product cloned into a GFP Vector, the complex transfect into the Sheep fibroblasts. screening positive cloning by use of G418, The expressed products of rap-1gene were identified by western blotting, The resμlts revealed that rap-1gene had been expressed successfμlly. This studies was the basis for studying the higher structure of the rap-1protein, and provided structural basis for researching the interaction between the protein and the host cell, which will be the basis of the target medicine.