犬脾细胞白介素-18(IL-18)基因的克隆与序列分析

Cloning and sequencing interleukin-18 from dog spleen

为了研究白细胞介素_1 8(IL_1 8)的活性和功能 ,将犬脾细胞经ConA诱导 ,用TRIzol裂解 ,提取总RNA ,通过反转录聚合酶链反应 (RT_PCR)扩增出犬IL_1 8的cDNA ,然后连接到pMD1 8_T载体上 ,转化DH5α感受态细胞 ,获得阳性重组质粒。核苷酸序列结果表明 ,我们得到了长 686bp的基因 ,含有一个 5 82bp的开放阅读框架 ,编码 1 93个氨基酸。与已知犬序列同源性可高达 99.6% ,与已知猪序列的同源性为 89.8% ,所克隆基因为今后进一步研究IL_1 8基因的功能奠定了基础。

To provide a basis for further investigation of the function of dog IL_18,a pair of primers specific to dog Interleukin_18 (IL_18)were designed and synthesized according to the known sequence from Genbank, and were used to amplify dog IL_18 cDNA by RT_PCR from total RNA of dog spleen cells induced with ConA. RT_PCR product was cloned into pMD18_T vector. The nucleotide acid sequence had been determined after being identified by restriction endonucleases digestion. The result showed that cloned IL_18 cDNA was 686 bp in length with a 582 bp open read frame encode a polypeptide of 193 amino acid.Compared with published dog IL_18, nucleotide acid and amino acid homology are 99.6 % and 99.8 % respectively. Cloned dog IL_18 showed 89.8 % homology with pig IL_18.