Establishment Of Detection Methods Based On Spherical Body Protein 3(SBP3) Gene Of Ovine Babesia

Babesia is widely distributed in temperate,tropical and subtropical regions of the world,causing serious damage to human and animal health.Babesia is different from Toxoplasma gondii,Plasmodium and other Apicomplexa protozoa.There are Microneme,Rhoptry and Spherical bodies in the apical complex of Babesia,but no Dense granules are found.In present research,the full-length sequences of SBP3 gene was amplified from the genomic DNA and c DNA of six Chinese ovine Babesia strains.Gene sequence alignment,analysis of structural characteristics and genetic evolution were carried out to understand the molecular characteristics of the SBP3 genes and taxonomic relationship of the Chinese ovine Babesia strains.SBP3 gene was used as diagnostic marker gene to establish real time PCR detection method,nested PCR detection method and High-resolution melting analysis method,respectively,to meet different detection purposes.The recombinant protein of SBP3 was obtained by prokaryotic expression system,and its polyclonal antibody was prepared,which provided biological materials for evaluating the potential of SBP3 as a candidate antigen for immunological detection.The main research contents and results are as follows:1.The full-length sequence of SBP3 gene was amplified from genomic DNA and c DNA of six Chinese ovine Babesia strains;The sequence alignment,structural feature and phylogenetic analysis were performed using bioinformatics techniques.The results showed that there was no intron in the SBP3 gene in Chinese ovine Babesia.The full length sequences of SBP3 genes in Babesia sp.Xingjiang/Dunhuang,Babesia motasi Ningxian/Hebei and Babesia motasi Lintan/Tianzhu were 3 195 bp,3 351 bp and 3 348 bp,respectively.The phylogenetic analysis showed that the six Chinese ovine Babesia were divided into two species,and there might have two subspecies of Babesia motasi.2.The SBP3 gene of six Chinese ovine Babesia strains was used as diagnostic marker gene.Through sequence analysis,specific primers were designed to successfully establish the fluorescent quantitative PCR for the detection of six Chinese ovine Babesia strains,the nested PCR for the differential detection of two kinds of Chinese ovine Babesia,and the High-resolution melting analysis method for the differential detection of two species of Chinese ovine Babesia and two subspecies of Babesia motasi.The three molecular detection methods had good specificity and sensitivity,and there was no cross-reaction between the three molecular biological methods and Theileria luwenshuni,Theileria uilenbergi and Anaplasma ovis.The minimum detection limitation of these methods was 0.5 pg-10 pg of ovine Babesia genomic DNA.3.Primers were designed to amplify the SBP3 gene fragment without signal peptide sequence using the SBP3 gene sequence of Babesia sp.Xinjiang as the template.The prokaryotic expression vector p ET-28a-BXJSBP3 was constructed and transformed into E.coli BL21 for induction expression and SDSPAGE analysis.The results showed that the 130 k Da recombinant protein(r BXJSBP3)was successfully expressed,and there was no significant difference in the expression level at 2,4,6 and 8 hours.The soluble analysis of bacterial precipitation ultrasonic fragmentation showed that the recombinant protein was mainly expressed in the form of inclusion bodies.r BXJSBP3 was purified and its concentration was determined to obtain soluble r BXJSBP3 with a concentration of 200 μg/m L.The purified r BXJSBP3 was used to immunize the experimental rabbits to prepare polyclonal antibodies.The polyclonal antibodies were analyzed by Western blot,and it was found that they could specifically recognize r BXJSBP3 and the natural SBP3 protein of merozoites of Babesia sp.Xinjiang.This study provided biological materials for further evaluation of the potential and function of SBP3 as a candidate antigen for immunological detection.