兔白细胞介素-15的克隆与表达

Cloning and expression of rabbit interleukin-15.

作者信息

Xiong Changyun, Hixson Patricia M, Mendoza Leonardo H, Smith C Wayne

机构信息

National Cancer Institute-Frederick, National Institutes of Health, Frederick, MD, USA.

出版信息

Vet Immunol Immunopathol. 2005 Aug 15;107(1-2):131-41. doi: 10.1016/j.vetimm.2005.04.013.

DOI:10.1016/j.vetimm.2005.04.013

PMID:15978671

Abstract

In order to understand the inflammatory mechanisms related to rabbit interleukin-15 (RIL-15), we cloned and expressed RIL-15 cDNA gene. A cDNA encoding RIL-15 was cloned from heart mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using hIL-15 primers. The RIL-15 cDNA contains an open reading frame (ORF) of 162 amino acids (aa) with a 48 aa leader sequence. The predicted molecular weight of the encoded protein (12.5 kDa) matched the size of recombinant IL-15 on Western blotting in an Escherichia coli (pET32a) expression system. Amino acid and nucleotide sequence analyses of RIL-15 revealed 82.7% and 87% homology with human IL-15 (hIL-15), respectively. RIL-15 is similar to the hIL-15 (hIL-15) in that it contains seven cysteine residues. RT-PCR showed that IL-15 is expressed in many tissues in the rabbit, including heart, spleen, lung, liver, muscle and kidney. Expressed and purified recombinant RIL-15, in the absence of the 48 aa leader sequence, stimulated the proliferation of cells of the mouse T cell line, CTLL-2, and its activity is comparable to hIL-15. Western blotting demonstrated that recombinant RIL-15 can be recognized by anti-IL-15 neutralization antibody. Western blotting also confirmed that IL-15 is present in many tissues including heart, spleen, lung, liver, muscle and kidney.

摘要

为了了解与兔白细胞介素-15（RIL-15）相关的炎症机制，我们克隆并表达了RIL-15 cDNA基因。使用人白细胞介素-15（hIL-15）引物，通过逆转录聚合酶链反应（RT-PCR）扩增从心脏mRNA中克隆出编码RIL-15的cDNA。RIL-15 cDNA包含一个162个氨基酸（aa）的开放阅读框（ORF），带有一个48 aa的前导序列。在大肠杆菌（pET32a）表达系统中，经蛋白质印迹法检测，所编码蛋白质（12.5 kDa）的预测分子量与重组白细胞介素-15的大小相符。对RIL-15的氨基酸和核苷酸序列分析显示，其与hIL-15的同源性分别为82.7%和87%。RIL-15与hIL-15相似，都含有7个半胱氨酸残基。RT-PCR显示，白细胞介素-15在兔的许多组织中表达，包括心脏、脾脏、肺、肝脏、肌肉和肾脏。表达并纯化的重组RIL-15，在没有48 aa前导序列的情况下，刺激了小鼠T细胞系CTLL-2细胞的增殖，其活性与hIL-15相当。蛋白质印迹法表明，重组RIL-15可被抗白细胞介素-15中和抗体识别。蛋白质印迹法还证实，白细胞介素-15存在于包括心脏、脾脏、肺、肝脏、肌肉和肾脏在内的许多组织中。