洛阳土种鸡IL-2 cDNA的克隆及原核表达

Cloning and Prokaryotic Expression of IL-2 Gene of Luoyang Local Chicken in E.coli

应用RT-PCR技术从新城疫I系疫苗诱导的洛阳土种鸡胚脾淋巴细胞中扩增到全长0.43kb的鸡IL-2基因cDNA,将IL-2cDNA克隆到PMD-T载体,测序结果表明,该基因为不含终止密码子的目的基因。将该目的基因克隆到原核表达载体PET-28a,获得的重组质粒PET-28a-IL2经酶切、PCR及序列测定,表明IL-2基因插入的位点、大小与读码框均正确。PET-28a-IL2在大肠杆菌BL21(DE3)中经IPTG诱导,表达产物经SDS-PAGE电泳分析,在14ku处出现一条约占总蛋白21%的蛋白带,表明所克隆的IL-2基因在大肠杆菌中得到良好的表达,为进一步研究IL-2的生物学特性奠定了基础。

A cDNA encoding Chicken interleukin-2（IL-2）was amplified by reverse transcription-polymerase chain reaction （RT-PCR） from Luoyang local Chicken embryo spleenocytes . Sequences analysis showed that the target gene was Chicken IL-2 and no termination codon. The IL-2 gene was inserted into the bacterial plasmid PET-28a, resulting in the construction of PET-28a-IL2 prokaryotic expression plasmids. The recombiant plasmid PET-28a-IL2 containing IL-2 gene were identified by restriction enzymes analysis and PCR method and sequenced to confirm its rightness. The recombinant fusion protein was highly expression in E. coli BL21（DE3）four hours later induced by IPTG and the molecular weight is about 14 ku. These provide a basis for the study on the biological property of chicken IL-2.