Development Of The Real-time PCR For Bovine-derived CD300a Gene And Construction Of Eukaryotic Expression Vector

CD300a is an important member of the CD300 superfamily.It is one of the surface antigens of leukocytes and plays an important role in the regulation of different immune cells.CD300a is widely expressed in myeloid cells and lymphoid cells,and the difference in expression depends on the type of cells,and is involved in regulating the growth of cells,and is closely related to diseases such as allergic diseases,infectious diseases and chronic inflammation.In this study,a pair of specific primers was designed based on the CD300a gene sequence of bovine source in GenBank.The CD300a gene fragment was amplified by cDNA of bovine mononuclear macrophage,and the SYBR Green I fluorescence quantitative PCR method for detecting the gene was established,and the standard curve of fluorescence quantitative PCR was made.nother pair of primers with enzyme cut site,amplified CD300a whole gene,constructed the eukaryotic expression vector of CD300a whole gene,and transfected in HEK-293T cell.The results showed that the CD300a gene SYBR Green I fluorescence quantitative PCR detection method was successfully established in this study.The sensitivity was 10 copies/μL;the amplification efficiency E=90.0%of the quantitative PCR standard curve was E=90.0%,and the correlation coefficient R₂=0.999,indicating that this method could be used to detect the expression of bovine CD300a in the cells.Cloning and sequencing of the whole gene of bovine CD300a found that the total length of the gene was 900bp,containing a transmembrane spiral domain,182-204 in the amino acid sequence,1-181 in the outer region of the membrane and 205-300 in the inner membrane region,and the prediction of the signal peptide shear site at the twenty-fifth amino acid residues.In addition,the eukaryotic expression vector pEGFP-CD300a was successfully constructed and transfected into HEK-293T cells through liposomes,which proved that CD300a protein was successfully expressed in HEK-293T cells.The above results indicate that the research results provide a basis for studying the role of bovine CD300a in immune regulation.