摘要
根据GenBank中登录的鸭IL-2基因序列设计并合成了1对特异性引物,以经ConA诱导的广州鸭外周血淋巴细胞提取的总RNA为模板,用RT-PCR方法扩增出长度为360 bp的目的基因片段,并将该基因克隆到pMD18-T载体上。酶切鉴定、PCR鉴定及序列测定结果表明,获得了鸭IL-2成熟蛋白基因的完整克隆。测序结果表明,该成熟蛋白基因由360个核苷酸组成,共编码119个氨基酸。克隆的鸭IL-2基因与GenBank上序列号为AY173028及AF294322的鸭IL-2基因的同源性高达99.4%。将目的基因克隆至真核表达载体pPICZαC上,构建了重组质粒pPICZαC-DuIL-2。酵母转化子经甲醇诱导发酵分泌表达了鸭IL-2基因。SDS-PAGE分析证实,鸭IL-2基因在毕赤酵母(Pichia pastoris)中表达成功,表达的重组蛋白的分子质量约为14.3 ku。
Based on the sequence of interleukin-2（IL-2） gene in GenBank,a pair of primers was designed to amplify the IL-2 mature protein gene from Guangzhou duck（DuIL-2） by RT-PCR with the total RNA prepared from peripheral blood lymphocyte induced by ConA as templates. The RT-PCR product was approximately 360 bp in size. The DuIL-2 mature protein gene was cloned into the pMD18-T simple vector. Identification by enzymatic digestion, PCR amplification and sequencing indicated that the DuIL-2 mature protein gene was cloned successfully. The result demonstrated that the cloned gene was consisted of 360 nucleotides,encoding 119 amino acids. The cloned gene shared 99.4% identity with those ones available in GenBank （AY173028 and AF294322）. The target gene was Cloned into pPICZaC vector,and the recombinant plasmid pPICZaC-DuIL-2 was constructed. The expression of rDuIL-2 was induced with met-hanoi. SDS-PAGE analysis showed that the rDuIL-2 protein was secreted successfully in the culture medium by Pichia pastoris strain X33 and it was 14.3 ku in molecular mass.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第3期229-232,共4页
Chinese Veterinary Science
