貉IL-2基因的克隆与进化关系的分析

根据GenBank上已发表的犬科动物的白细胞介素-2（IL-2）基因序列，在开放阅读框架上下游的保守区域设计了1对引物。无菌采取貉血，使用淋巴细胞分离液分离外周血液中的淋巴细胞，加入ConA，于二氧化碳培养箱中培养48h后收集培养细胞。以培养细胞中提取的总RNA为模板，应用RT—PCR方法，扩增出貉的IL-2基因。分离纯化片段，连接T载体转化大肠杆菌并测序。结果表明：IL-2基因开放阅读框架全长486bp，编码155个氨基酸。该序列与大、狐等犬科动物的IL-2基因同源关系最近，与大熊猫、家猫的IL-2基因同源关系相对较近，与人、马、牛、绵羊的IL-2基因有一定的遗传距离，与禽类鸡的IL-2基因同源关系最远。

Cloning and Genetic Phylogeny of the Raccoon Dog Interleukin-2 Gene

According to the nucleotide sequence of canine Interlenkin-2 published in Genbank,a pair of primers were designed in high homology regions of open reading frame(ORF) of raccoon dog.Blood of raccoon dog was collected by aseptic operation.Peripheral blood mononuclear cells(PBMC) were separated by lymphocyte separating medium and cultured in RPMI1640 medium for 48 hours after adding conA.Then interlenkin-2 gene was cloned from the concanvalin stimulated peripheral blood mononuclear cell(PBMC).DNA sequence fragment was purified, cloned into T vector and transformed into Escherichia coli.The results indicated that the ORF of raccoon dog IL-2 gene was 486 bp nucleotides in length,which encoded 155 amino acids.Sequence analysis showed that the sequence was closest homologous with IL-2 gene of dog,fox and the other canids,and was relatively close homologous with that of giant panda and cat.The relation between IL-2 gene of the animal with that of people,horses,cattle,sheep had a certain genetic distance,and farthest approached to that of chicken.