| Babesia orientalis is a newly discovered and confirmed new species of Babesia bovis in recent years. Clinical studies have shown that it is only pathogenic to buffalo and is transmitted by egg from Rhipicephalus haemaphysaloides. It distributes in the south of the Changjiang River region. The clinical manifestations of diseased animals is anemia,fever, jaundice and hemoglobinuria and other symptoms, may lead to death, thus southern China’s agricultural production and aquaculture industry suffered a serious threat. At present, prevention and treatment of Babesia buffalo disease are still in the original drug-bath stage,which can not work out well; the vaccines of Babesia oriental disease control are rarely reported, and the key molecules involved in the invasion of parsite during host infection are also lack of systematic research.Based on this study, we selected the key invasive TRAP protein, which was identified in the Apicomplex protozoa, to capture the Babesia orientalis Thrombospondin-related anonymous protein 2 from the existing Babesia orientalis gene bank （BoTRAP2）. This gene was cloned and analyzed, and the interaction between BoTRAP2 protein and buffalo erythrocyte membrane protein was explored. The functional characteristization of BoTRAP2 protein and its domain in binding to heparin were analyzed. And finally the form of BoTRAP2 protein crystals was also attempted.（1） Cloning expression and bioinformatics analysis of TRAP2 gene in Babesia orientalisA sequence of highly homologous sequences of the TRAP2 gene sequence（XM001609762.1） was found in the genebank of the Babesia orientalis by multiple sequence alignment. A 2817bp fragment was amplified from the gene library of Babesia orientalis, and the nucleotide sequence was analyzed by bioinformatics. It was found that it contained two vWFA domain, two TSP domain, one domain of the MIADS region, one domain across the membrane structure, and the typical TRAP-like family of nucleotide structure is consistent, indicating that the gene sequence for the Babesia orientalis TRAP2 gene. The homology analysis showed that the amino acid sequence is 94%homologous to the TRAP2 amino acid sequence of Babesia bovis and the bioinformatic software analysis showed that the protein has good immunogenicity.（2） Interaction between TRAP2 protein and erythrocyte membrane protein in Babesia orientalisThe expression of TRAP2-vWFA-TSP was designed by using the primers cloned into the key domain of the TRAP2 protein （containing vWFA, TSP, MIADS key sequence region）. The prokaryotic expression plasmid containing the single His tag was constructed PET-28a-TRAP2-vWFA-TSP was transferred to BL21 expressing strain. Far-western validation of recombinant TRAP2-vWFA-TSP protein interacts with erythrocyte membrane protein. The results showed that there is a suspected target band on the erythrocyte membrane protein, indicating that the TRAP2 protein and the erythrocyte membrane protein the combination of the role.（3） Adhesion bonding of TRAP2 protein and its domain between with heparinThe recombinant plasmids of pET-28a-TRAP2-vWFA-TSP, pET-28a-TRAP2-vWFA and pET-28a-TRAP2-TSP were constructed into BL21 cells and explored to obtain a large amount of recombinant TRAP2- VWFA-TSP, TRAP2-vWFA, TRAP2-TSP protein, and the interaction between TRAP2 protein and its domain between with heparin was verified by ELISA. The results showed that TRAP2 protein and its domain had a good binding to heparin, and the binding affinity of TRAP2-vWFA-TSP protein to heparin was stronger than that of TRAP2-vWFA , TRAP2-TSP alone. It also indicates that the TRAP2 protein may play a synergistic role with its single domain when combined with heparin.（4） The crystal screening of TRAP2 protein in Babesia orientalisThe purified pET-28a-TRAP2-vWFA-TSP protein was purified with the GE Superdx 200pg molecular sieve purification column. The pET-28a-TRAP2-vWFA-TSP protein was digested with the dimer complex. In the form of the existence of the molecular purification can be obtained after the concentration of 9mg/mL protein, purity> 90% of the target protein. Through the commercial crystallization board to obtain a suspected protein crystal small crystal block, by changing buffer Salt ions, the concentration of precipitants to optimize, the experiment did not get the desired results, there is need for further exploration of the conditions.In this study, TRAP2 gene was successfully cloned and analyzed by bioinformatic tools. The key domain of TRAP2 gene was expressed by prokaryotic expression system,and its immunogenicity is confirmed. The TRAP2 protein and its domain protein were confirmed to be interacted with heparin specifically. These results are of great significance for the further study of the molecular mechanism of infestation and the prevention of Babesia. |
