大熊猫IL-2基因的克隆与序列分析

Cloning and Sequence Analysis of IL-2 Gene in Ailuropoda Melanoleuca

本实验应用反转录-聚合酶链反应(RT-PCR)技术,从ConA诱导培养的大熊猫外周血淋巴细胞总RNA中扩增得到大熊猫IL-2基因,并将其克隆到PGEM-T载体中.经菌落PCR鉴定、序列测定及序列分析,结果表明,经克隆得到的IL-2基因开放阅读框由465个核苷酸组成,编码一个由155个氨基酸组成的多肽,包括编码20个氨基酸的信号肽和135个氨基酸的成熟肽.该基因(已在Genbank中登录,序列号为:DQ852339)与已知的其他哺乳动物如犬、猫、人、猪、牛、羊、马、兔、鼠等的IL-2核苷酸同源性在66.5%(鼠)—91.5%(犬)之间;IL-2编码氨基酸同源性在54.8%(鼠)—85.2%(犬)之间;进化树构建结果表明大熊猫与犬亲缘关系最近.

By reverse transcription polymerrase chain reaction（RT-PCR） , the giant panda IL-2 gene was cloned from total RNA which was extracted from ConA-stimulated-cuhured peripheral blood lymphocytes of the giant panda and the amplified products of PCR were cloned into PGEM-T vector. After identification and sequencing, the sequence analysis revealed that the open reading frame of the giant panda IL-2 gene contained 465 nucleotides which encode 155 amino acids,the signal peptide contained 20 amino acids and the mature IL-2 gene was composed of 135 amino acids. Compared with other known mammals in Genbank, including dog, cat, human, pig, cow, horse, goat, rabbit, rat and so on, the homology of nucleotide sequence was between 66.5 % （rat） and 91.5 % （ dog）, the homology of amino acid sequence was between 54.8 % （rat） and 85.2 % （dog）. Phylogenetic analysis based on the nucleotide sequence of the IL-2 gene showed that the giant panda had the most closed genetic relation with the dog.