Cloning, Expression And Analysis Glycophorins Of Bovine Erythrocytes And Babesia Orientalis SBP3

Babesia orientalis is a tick-borne hemoprotozoan parasite and transmitted by Rhipicephlus haemaphysaloides transovarially with the infected stage being the adult tick, is the causative agent of babesiosis in buffalo. B.orientalis is confirmed to be an independent new species in previous study in our laboratory. B.orientalis only infecting buffalo rather than cattle or dairy cattle, which is speculated that the reason is probably due to the difference of Babesia invasion ligands or bovine erythrocyte receptors. But the molecular process of erythrocyte invasion by B.orientalis merozoite is unclear, to analysised reasons of B.orientalis only infecting buffalo in molecular level, we study on bovine rerythrocyte receptors and B.orientalis ligands.1. Cloning, expression and analysis glycophorins of bovine erythrocytes(1) Cloning and analysis the sequence of GYPC from cattle, dairy cattle and buffaloThe four, two, six pair pecificity primers were designed through bovine GYPC, DARC, GYPA gene sequence released in GenBank. GYPC, DARC, GYPA gene was obtained by PCR with whole blood gene template from cattle, dairy cattle and buffalo. The PCR products were inserted into clonging vector and identificated by sequencing. Four exon of GYPC, two exon of DARC, six exon of GYPA were spliced by the clustalx and Primer Premier5.0. The hydrophilicity, antigenicity, surface activity, isoelectric point, primary structure and secondary structure of GYPC, DARC, GYPA from cattle, dary cattle and buffalo were predicted by the DNAStar software. Transmembrane domains, glycosylation sites and signal peptide were predicted by online software and comparative analysis the differences among cattle, dary cattle and buffalo. The results showed that GYPC, GYPA from the buffalo have large differences with cattle and dairy cattle in the primary structure, secondary structure, isoelectric point, glycosylation sites and the number, the DARC from cattle, dairy cattle and buffalo are vary in the primary structure, secondary structure, isoelectric point, glycosylation sites and the number.(2) Eukaryotic expression of cattle GYPA geneThe cloned GYPA gene was recombined into eucaryotic expression plasmid pcDNA3.1(+) connecting with the green fluorescent protein gene EGFP. The Eucaryotic expression plasmid pcDNA3.1(+)-GYPA-EGFP was established after identification by sequencing. pcDNA3.1(+)-GYPA-EGFP was transfercted into PK cell and MDBK cells by liposomes to express cattle GYPA gene transiently. After transfection of24hours, the green fluorescent were observed, and the fluorescence intensity went up after36h. The fluorescence of blank control plasmid pcDNA3.1(+)-EGFP was distributed in the intracellular. The fluorescence of pcDNA3.1(+)-GYPA-EGFP was distribution around the cell membrane and the fluorescence intensity was weaker than the blank. The GYPA gene contains a signal peptide and the target protein was secreted into the cell surface, which reduced the intensity of the fluorescent protein.In short, GYPC, DARC, GYPA from cattle, dairy cattle and buffalo were cloned and comparative analysised. It is speculated that the differences of GYPC and GYPA gene sequences among cattle, cattle dairy and buffalo may the reason of B. orientalis only infecting buffalo. These results presented that GYPC and GYPA gene provided basic theory for further research.2.Study on cloning and expression of SBP3gene of B. orientalis and immunorectivi-ty of its recombinant protein(1) The B.orientalis SBP3gene cloning and analyzing.The specificity primer was designed through B.orientalis genome sequencing results in our laboratory and B.bovis SBP3gene published in GenBank. B. orientalis SBP3gene was obtained by PCR with whole blood gene template from a sick cattle. The results showed that B.orientalis SBP3gene is an open reading frame with3237bp, according to which its molecular weight was deduced to be118kDa with1079amino acid residues. The SBP3sequence of B.orientalis and B.bovis were analyzed by gene sequence homology online, the highest homology is70%. B.orientalis SBP3nucleotide sequence lost33bp and the amino acid sequence lost11AA, which indieated that B.orientalis was a new species of Babesia family. Antigenicity prediction of the B. orientalis SBP3gene indicate that it have good antigenicity.(2) Prokaryotic expression of B.orientalis SBP3gene and its immunoreactivity study.SBP3gene were divided into three respectively927bp,1329bp,1026bp named SBP3-1, SBP3-2, SBP3-3. Then they were inserted into prokaryotic expression vector pET-28a(+), prokaryotic expression plasmid pET-28a(+)-SBP3-1, pET-28a(+)-SBP3-2, pET-28a(+)-SBP3-3were established, and they were transformed into BL21-CondonPlus, multicopy recombinant strain induced to express with IPTG. The40kD,53kD,42kD protein were detected in Western-blot, which is match with the theory size. The B. orientalis SBP3recombinant protein react specifically with natural antibodies in positive bovine serum, the B. orientalis SBP3has good immunogenicity.In short, B.orientalis SBP3gene was cloned, expressed and western blot in this study. All the results supports that SBP3protein can be used to be a stable diagnosis instrument for bovine babesiosis detection, as well as a candidate material for producing bovine babesiosis genetic engineering subunit.