猪IL-2基因的克隆、序列分析及其杆状病毒表达载体的构建  

Cloning,genetic analysis and baculovirus expression vector construction of porcine interleukin-2

作　　者：郭小参[1,2] 崔保安[1,2] 陈红英[1,2] 王彦彬[1,2] 方剑玉[3] 吕晓丽[1,2] 王东方[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002 [3]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医学报》2009年第2期129-133,共5页Chinese Journal of Veterinary Science

基　　金：国家"十五"食品安全重大攻关专项(2001BA804A30-11)

摘　　要：参照GenBank发表的猪IL-2 cDNA基因序列设计1对引物,将猪脾淋巴细胞在伴刀豆球蛋白A(Con A)的刺激下体外培养27 h后,提取激活淋巴细胞总RNA,进行反转录-聚合酶链反应(RT-PCR)扩增,克隆到pGEM-TEasy载体上并测序。测序结果显示,克隆的猪IL-2 cDNA全长为516 bp,开放阅读框(ORF)包含465 bp,编码154个氨基酸,相对分子质量为17 400,等电点为5.27,此cDNA与已报道的猪IL-2同源性为100%,与猫、牛、鸡、犬、鸭、山羊、马、人、家鼠等的IL-2基因进行比较分析,核苷酸同源性分别为83.7%、82.6%、28.2%、80.6%、29.6%、83.4%、81.3%、82.0%和61.5%。将此IL-2基因亚克隆到杆状病毒转移载体pFastBacDual后获得了重组质粒pFBD-IL2,进而转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,发生转座作用;经抗性及蓝白斑筛选得到了含猪IL-2基因的重组DNA,将其命名为Bacmid-IL2。本试验为进一步在昆虫细胞中表达猪IL-2基因、开发研制新型免疫佐剂奠定了基础。One pair of primers was designed according to porcine IL-2 gene sequences published in GenBank. The interleukin-2 cDNA of porcine was amplified by RT-PCR from total RNA extracted from spleen lymphocytes stimulated with 10 mg/L ConA in vitro for 27 hours. Then the amplified eDNA was cloned into pGEM-T Easy vector and sequenced. The result showed that the full length of the porcine IL-2 was 516 bp （ORF was 465 bp ）, encoding a peptide of 154 amino acids whose molecular is 17 400,pI is 5.27. The sequence was just the same as GenBank published. Compared of nhe nucleotides sequence of porcine IL-2 with those of cat, cattle, chicken, dog, duck, goat, horse, human and house mouse. The result showed that the nucleotides homologies were 83.7% ,82.6% ,28.2% ,80.6%, 29.6% ,83.4% ,81.3% ,82.0% and 61.5%, respectively. The baculovirus transfer vector of pFastBaeDual containing pIL-2 was constructed and transformed into DH10Bac which contains a shuttle vector of bacmid. The study paved the way of the expression in insect cell and the development of new immunologic adjuvant.

关 键 词：猪白细胞介素-2 克隆 序列分析 杆状病毒 

分 类 号：S852.4[农业科学—基础兽医学]