恒河猴XC趋化因子受体1(XCR1)基因的克隆和体外表达

Cloning and in vitro expression of XCR1 of Rhesus macaque

目的了解恒河猴XC趋化因子受体1(XCR1)基因特征,并在HEK293T细胞上表达。方法采用反转录PCR和cDNA末端快速扩增法(RACE)克隆恒河猴XCR1基因、并与GenBank中其他物种XCR1的cDNA序列进行比对。构建真核表达载体3.1-XCR1并转染HEK293T细胞,采用流式细胞术、Western blot法和激光扫描共聚焦显微镜技术检测XCR1蛋白的表达。结果恒河猴XCR1 cDNA序列全长1665 bp,包含415 bp的5'非翻译区(5'UTR)、1003 bp的编码区和248 bp的3'UTR;恒河猴XCR1编码区氨基酸序列与人XCR1的相似性为96.8%,而且具有相似的结构特征:含有七次跨膜结构,酸性N端以及保守的G蛋白锚定功能性基序HRYLSVV。与基因组序列比对和跨外显子反转录PCR结果表明,在恒河猴多种组织中只存在缺失第二外显子的剪接变异体。恒河猴XCR1可在HEK293T细胞中表达Mr40 000左右的分子、主要分布于细胞质和细胞膜上。结论除mRNA剪接异构体以外,恒河猴XCR1分子与人高度相似,并且能在HEK293T细胞中有效表达。

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 XCR] 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 XCR 1 gene of Rhesus macaque is highly similar to that of human except mRNA transcript variant, and XCR1 could be expressed in HEK293T cells effectively.