摘要
应用反转录—聚合酶链式反应(RT-PCR)技术,在国内首次从ConA诱导培养的长毛兔和獭兔外周血淋巴细胞总RNA中扩增出IL-4基因。将扩增基因克隆入pMD18-T载体,经序列测定分析表明,长毛兔和獭兔IL-4基因全长均为441bp,两序列间碱基组成无差异(登录号:EF606761和EU639687)。长毛兔和獭兔IL-4基因编码147个氨基酸,其中前24个氨基酸组成信号肽,余下的123个氨基酸组成成熟肽。与已报道的犬、猫、人、猪、牛、羊、马、大熊猫和鼠的IL-4核苷酸同源性分别为61.8%、63.4%、69.8%、65.1%、64.1%、64.1%、63.1%、57.0%、60.5%,推导的氨基酸同源性分别为47.2%、50.0%、53.6%、52.1%、48.3%、46.9%、52.1%、43.7%、43.0%。将测序正确的阳性质粒采用脂质体法转染COS-7,用RT-PCR鉴定转染细胞中IL-4基因的表达,并通过MTT法检测到表达的IL-4蛋白具有一定的生物学活性。
Rabbit interleukin-4 cDNA from lymphocyte was synthesized by reverse transcription polymerase chain reaction（RT-PCR） from mRNA extracted from blood lymphocytes of Angola and Rex rabbit,which were stimulated by 10 μg/mL Con A in vitro.The interleukin-4 cDNA was cloned into pMD18-T vector for sequence.The full cDNA length of the IL-4 gene was 441 bp in Angola and Rex rabbits.The IL-4 encoded a peptide of 147 amino acids,containing signal peptide composed of 24 amino acids and mature interleukin-4 gene composed of 123 amino acids. The Angola and Rex rabbits had 61. 8%, 63. 4%,69. 8%, 65.1%, 64.1%, 64. 1%. 63.1%, 57.0% and 60.5%; identity with the 11. 4 genes of dog, cat, numan, pig, cow, horse, goat, panda and rat, respectively. The deduced amino acid had 47.2%, 50.0%, 53. 6%, 52. 1%, 48.3%, 46.9%, 52.1%, 43.7% and 43.0% identity with the expressed IL-4 of dog, cat, human, pig, cow, goat, horse, panda and rat, respectively. The recombinant plasmid was transfected by lipofectin to COS-7. Expression of IL-4 gene was detected by RT PCR. The transient expression product has some biological activities analyzed by MTT.
出处
《中国畜牧兽医》
CAS
北大核心
2009年第11期51-56,共6页
China Animal Husbandry & Veterinary Medicine
基金
教育部<长江学者和创新团队发展计划>创新团队项目(IRTO848)
国家公益性行业(农业)科研专项项目(nyhyzx07-040)
国家农业科技成果转化资金项目(2007GBZF000277)
关键词
长毛兔
獭兔
克隆
真核表达
白细胞介素-4
Angola rabbit
Rex rabbit
cloning
eukaryotic expression
interleukin-4
