Cloning and in vitro expression of XCR1 of Rhesus macaque

: Objective To characterize the gene of Rhesus macaque XC chemokine receptor 1 (XCR1) and express it in HEK293T cells. Methods Rhesus macaque XCR1 gene was amplified by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA end (RACE). The sequence alignment was compared with XCR1 of other species in GenBank. Eukaryotic expression plasmid 3.1-XCR1 was constructed and transfected into HEK293T cells. The expression of XCR1 was identified by flow cytometry, Western blotting and confocal microscopy. Results The complete cDNA sequence of XCR1 was obtained, containing 415 bp 5'UTR, 1003 bp coding region and 248 bp 3'UTR. Amino acid alignments of Rhesus macaque XCR1 not only showed 96.8% identity with human, but also shared the same protein characteristics: containing seven transmembrane domains, acidic N-terminal and conserved G protein anchor functional motif HRYLSVV. The comparison with genome sequences and trans-exon RT-PCR revealed only one transcript variant with deletion of the second exon in various tissues of Rhesus macaque. In addition, Rhesus macaque XCR1 was successfully expressed in HEK293T cells, Mr was about 40 000 in size and primarily localized in the cytoplasm and on the cell surface. Conclusion The XCR1 gene of Rhesus macaque is highly similar to that of human except mRNA transcript variant, and XCR1 could be expressed in HEK293T cells effectively.