Functional Analyses Of BmGPI13 And BmGPI17 In The Process Of Erythrocyte Invasion By Babesia Microti

Babesia microti is an intraerythrocytic apicomplexan parasite.It is widely distributed among the world and can infect human beings and small rodents.Human infected with B.microti is often accompanied by hyperparasitemia,which can lead to death in severe cases.At present,no vaccine or drug has been developed to effectively prevent and treat B.microti infected.Glycosylphosphatidylinositol(GPI)anchored protein is an important secretory protein of the apicomplexa,which has been proved to play an important role in adhesion,anchoring and other important functions in the invasion of host cells,and commercially effective vaccines against such antigens are available clinically.Therefore,Bm GPI13 and Bm GPI17 were studied in this study,to explore the protein function in the invasion and to reveal the molecular mechanism of the invasion by B.microti.In this study,Bm GPI13 and Bm GPI17 genes were cloned,expressed and purified.The function of Bm GPI13 and Bm GPI17 genes were studied subsequently.And to be a potential vaccine candidate antigen was evaluated by antibody neutralization test.The main research work including:(1)Antigenicity analysis of Bm GPI13 and Bm GPI17First of all,using the c DNA of B.microti as template,we cloned the coding sequence of Bm GPI13 gene with a total length of 960 bp and the coding sequence of Bm GPI17 gene with a total length of 1371 bp.Then they were inserted into p GEX-6p-1 prokaryotic expression vector respectively,and the recombinant plasmids were transformed into E.coli Transetta for recombinant protein expression.The recombinant protein Bm GPI13-GST and Bm GPI17-GST were purified from supernatant and inclusion body respectively.Western blot analysis showed that both Bm GPI13-GST and Bm GPI17-GST proteins can be recognized by the positive sera infected by B.microti,proving that the recombinant protein has good reactivity.Next,the rabbit polyclonal antibody was prepared using recombinant protein.It was found that the polyclonal antibody can specifically recognize the specific antigen in the lysate of B.microti,and the natural protein sizes of Bm GPI13 and Bm GPI17 were about 37 k Da and 50 k Da respectively.(2)Identification of invasion function of Bm GPI13 and Bm GPI17It was found that Bm GPI13 did not have the ability to bind to the host RBC,while Bm GPI17 could bind to the host RBC.In the exploration of Bm GPI17 RBC binding domain,we found that its F3 region has the ability to bind to the host RBC,and the use of specific antibodies can block the binding between Bm GPI17-F3 and RBC,indicating that the binding between Bm GPI17 and RBC is specific.Far-western analysis showed that Bm GPI17 may interact with other B.microti proteins in the process of invasion by B.microti.The results of antibody neutralization test showed that the polyclonal antibodies against Bm GPI13 and Bm GPI17 were significantly different from those of the control group at a concentration of 1 mg/ml,indicating that they could inhibit the growth of B.microti.In conclusion,Bm GPI13 and Bm GPI17 genes were successfully cloned and expressed in this study.The functions of Bm GPI13 and Bm GPI17 were studied by erythrocyte binding assay,antibody blocking binding assay,Far-western test and antibody neutralization test.The functions of Bm GPI13 and Bm GPI17 in the process of invasion were determined,which provided a new scientific basis for exporing the invasion mechanism of B.microti and the development of related vaccines.