摘要
根据GenBank发表的鸭白细胞介素-2(IL-2)基因序列设计并合成了特异性引物,以经ConA诱导的广州白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.将该基因克隆到pMD18-Tsimple vector上,经酶切、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.构建的表达质粒pET-IL-2经BamHI、XhoⅠ双酶切鉴定构建正确;经SDS-PAGE分析,在约33 000处出现新的蛋白条带,Western-blotting分析表明,融合蛋白能够被抗His单克隆抗体识别,体外活性检测表明,重组蛋白具有促进淋巴细胞增殖的活性.
Based on the sequence of gene IL-2 available in GenBank^TM, A pair of primers was designed and subsequently a fragment of 360 bp was amplified by RT-PCR with the total RNA as template which isolated from peripheral blood lymphocyte of Guangzhou White duck and induced by ConA. The DuIL-2 maturation protein gene was cloned into pMD18-T simple vector, identified by enzymatic digestion and confirmed by PCR and sequencing. The results indicated that DulL-2 maturation protein gene was cloned successfully. The expression vector pET-IL-2 was constructed and verified by digestion with BamHI and Xho I. After induction with IPTG, there was a specific protein band of aooroximatelv 33 000 on SDS-PAGE gel. Results of Western-blotting analysis indicated that the recombinant protein could react with monoclonal antibody against His. MTT colorimetric assay indicated that the recombinant protein could induce duck peripheral blood lymphocyte T lymphocytes in vitro.
出处
《华南农业大学学报》
CAS
CSCD
北大核心
2008年第4期83-86,共4页
Journal of South China Agricultural University
