Cloning And Primary Functionality Research Of Babesia Orientalis Immunological Related Protein

Babesia orientalis is an important intraerythrocytic protozoan parasite which transmitted by Rhipicephalus haemaphysaloides haemaphysaloides.It causes buffalo babesiosis which is one of the most important diseases of buffalo in central and south of China and is characterized by apathy,fever,anemia,icterus,haemoglobinuria and in some cases death.The proofs of morphology,life cycle,vector and molecular taxonomy were indicated that B.orientalis was a new species of Babeisa family.The previously constructed B.orientalis cDNA library was screened by McAb and PcAb in this study. Complete nucleotide sequence of positive clones were analyed and assembled using DNA Starver.6.0 software,and aligned.And then BoP 29 which was screened by McAb was cloned and expressed in E.coli.BoP29 was tested by western blotting and indirect immunofluorescence assay.An indirect enzyme-linked immunosorbent assay(ELISA) was developed using the rBoP29 as antigen.Analysis of 132 sera samples collected from buffaloes showed that there was no significant difference in relative effectiveness of rBoP29-ELISA and PCR.(1) Preparation of B.orientalis McAbAfter immunization of Balb/C mice with the B.orientalis solubility antigen,the stimulated splenocytes were routinely fused with SP2/0 myeloma cells to produce hybridomas.The positive clone cell strains were selected by BoMAg-ELISA and BoECSAg-ELISA.Eleven positive cell strains were abtained.They wereâ…£C₂A₇1G₁₀,â…£C₂A₇2E₂,â…£C₂A₇1F₆,â…£C₂A₇2G₄,â…£C₂A₇2C₅,â…£C₂A₇2F₁₀,â…£C₂A₇1E₃,â…£C₂A₇2F₂,â…¤C₉C₆A₂,â…¤C₉C₇B₁ andâ…¤C₉C₆C₁₁.(2) Immune screening of B.orientalis cDNA libarayB.orientalis cDNA libaray was screened by McAb and PcAb.B.orientalis protein BoP29 was obtained by using McAbâ…¤C₉C₆C₁₁,and another 8 B.orientalis proteins were screened out.They were ms-7-1,ms-37-1,ms-59-1,ms-70-2,ms-88-2,ms-99-1, ms-110-1 and ms-128-1.The BLAST and protein prediction results showed that BoP29 had a good antigenicity;ms-7-1,ms-37-1,ms-59-1,ms-99-1 and ms-110-1had a high siminarity with some protein of Babesia bovis;ms-88-2 had 34%homology with hypothetical protein Tb10.70.1650 of Trypanosoma brucei;ms-70-2 and ms-128-1 had no significant correlation information.(3) Clone and expression of BoP29A pair of oligonucleotide primers including the BamHI and SalI restrictionenzyme sites were designed to obtain the whole gene encoding rBoP29 from B.orientalis genomic DNA.The PCR product was cloned into the BamHI and SalI restriction enzyme sites of the pGEX-KG and pET-28(a) vector.The rBoP29 was expressed as a fusion protein in the E.coli BL21 according to the instructions.(4) Location and immunogenic of BoP 29To identify whether the rBoP29 had identical molecular weight and immunocompetence as we predicted,positive serum of B.orientalis was used to analyze the rBoP29 by SDS-PAGE and Western blotting.The Western blotting resulte showed that BoP29 had a good antigenicity as predicted.IFA assay result indicated that BoP29 was located at the menbrance of B.orientalis.(5) Development and appliction of His-BoP-29 indirect ELISAThe cutoff value of His-BoP-29 indirect ELISA was 0.25.One hundred and thirty two field buffalos' blood samples were collected from four places(Wuhan,Daye,Anshan and Jiayu) in Hubei province in China,and tested by indirect ELISA based on rBoP29 and semi-nested PCR.Thirty-nine(29.5%) positive samples for B.orientalis antibodies were detected by rBoP29-ELISA,while 44(33.3%) positive samples for B.orientalis DNA were found in the semi-nested PCR detected.The Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBoP29-ELISA and semi-nested PCR in identifying positive samples.In this research,the cDNA library was used to immune-sceening.Nine B.orientalis immunological related protein were obtained.This will lay foundations for further study on molecular biology and immunity,nosogenesis,prevention and control of the buffalo babesiosis.A novel bop29 gene of B.orientalis was identified by immunoscreening a cDNA library with one stain McAb and the gene product was used as antigen in ELISA to evaluate its immune reactivity.This implied that the BoP29 antigen is a candidate antigen for epidemiological survey and diagnosis of B.orientalis infection...