Genetic Screening?cloning And Expressing And Immunological Analysis Of Rhoptry Asscociated Protein Gene Of Babesia Microti

Babesia is obligate intracellular protozoa,important pathogens caused babesiosis of humans and animals.Tick is the predominant transmission vetor.Babesiosis is transmitted mainly through tick bite,blood transfusion and mother-fetus vertical transmission.They and other Piroplasma own conserved subcellular structures and invasion mechanism.Rhoptry-associated proteins,which released into the host cell,are considered to be the key molecules of invasion and replication of parasites in host cell and an immunosuppressive factor of the host cell mediated immunity in the stage of parasitophorous vacuole?PV?formation.Meanwhile,it indicated that Babesia microtic rhoptry-associated protein?BmRAP?can be used as a candidate vaccine and diagnostic antigen molecules in previous studies.Therefore,the study dedicated to clone and express the recombinant protein of BmRAP and immune performance analysis,which will lay the foundation for following study.Objective:This study aims to search the BmRAP gene sequences and the amino acid sequence through the screening of different databases;the C-BmRAP and N_BmRAP had been cloned and expression in vitron and got the purificated C-BmRAP and N-BmRAP;learn the immune capacity of the BmRAP through testing to verify the immune effect of the protein to lay the foundation for follow-up study.Method:Through screening NCBI,Uniprot,EMBL-EBI,GeneDB and PiroplasmaDB professional website,search the gene expressing rhoptry-associated protein of Babesia microti,and understand the basic physical and chemical properties and protein antigen epitope by bioinformatics analysis,to determine the truncation expression gene sequence.Then a restructuring of the carrier to clone and express recombinant BmRAP protein?rBmRAP?was built.The BALB/c mice were challenged with Babesia microti after immunization with purified rBmRAPs.The immune effects and possible mechanism of anti-infection about the rBmRAPs were explored by observing parasite density in thin blood smear and detecting the cytokines in sera.Results:1)Each BmRAP gene sequence of B.microti was respectively achieved from the EMBL-EBI and PiroplasmaDB database?the accession number XP₀12649548 and BBM_?04695?.Furthermore,after the APE software comparing,they are the same sequence,which consist of 4 443 bp and encod 1480 amino acids,with the molecular weight of 165.31 kDa.The protein contains a signal peptide,four structures of transmembrane?7-29/1256-1 278/1377-1399 and 1414-1433aa?.The BmRAP protein is probably a stable secreted protein containing better antigen epitopes.The phylogenetic analysis of BmRAP sequence revealed that the B.microti is closely related to the other species of Babesia,and distant to the parasites of the other genus.The homologies of the BmRAP sequence with that of B.bovis and B.divergens were 68%and 67%,respectively.2)Through bioinformatics analysis,the C-truncated and N-truncated protein gene sequences obtained for coding BmRAP were determined,and a restructuring of the vetor successfully was built,a higher purity of purified recombinant proteins as well as laid a theoretical foundation for the further study at the structure and function of the protein.3)The C-BmRAP and N-BmRAP group and normal control group performance results show that the N-BmRAP have the better immune protection.In the routine blood test results,there was no significant differences between groups of WBC?F = 1.842,P>0.05?;On the seventh day after infection,there have been obvious differences between groups among minimized the number of RBC and RBC's test results?F = 8.096,P<0.05?.4)The splenic sizes and weights of the each group of mice challenged with B.microti after immunization with recombinant proteins increased,and the spleen of the mice in the control group was obviously bigger than that in the C-BmRAP and N-BmRAP groups.At the seventh day after infection with B.microti,the expression quantity of TNF,IFN--?and IL-10 increased,IL-2,IL-4,IL-6 and IL-10 increased unsignificantly;the 21th day after infection,only the TNF has no obvious peak and the rest of the indicators is in peak condition.Conclusion:1)The achieved RAP of B.microti by searching in the professional database,containing more antigen epitopes,can be used as a candidate vaccine and diagnostic antigen molecules.2)It will lay a theoretical foundation for the further study at the structure and function of the protein.3)The immune protection of the C-BmRAP is better than the N-BmRAP.4)C-BmRAP and N-BmRAP can enhanced immune response.They can be used as the candidate antigen for vaccine.