犬IL-2基因克隆及原核表达

Cloning and Prokaryotic Expression of Canine IL-2 Gene

[目的]研究犬IL-2基因的克隆和表达,为开发新型免疫增强剂和基因工程疫苗提供理论支持。[方法]从犬全血中分离出白细胞,经刀豆素刺激培养20 h后,提取总RNA;根据GenBank中犬IL-2基因序列,设计1对引物,进行PCR扩增,并构建原核表达载体pET-28a,将其转化到宿主菌BL21中,经IPTG诱导表达,表达产物用SDS-PAGE进行分析。[结果]RT-PCR扩增出一条约500 bp的条带,与预期结果相符;重组质粒pMD18-T-IL2经双酶切后,产生一个约500 bp基因片段,说明目的基因被成功克隆;表达载体pET-28a-IL2经双酶切和PCR鉴定后,分别产生一个约500 bp目的片段,表明表达载体构建成功;表达产物经SDS-PAGE分析,分子量约为20 kDa,与预期蛋白大小基本相同。[结论]犬IL-2基因被成功克隆和表达。

[Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines.[Method] Leukocytes separated from canine whole blood were stimulated by concanavalin for 20 h,and then total RNA was extracted.According to the sequence of canine IL-2 gene published in the GenBank,a pair of primers was designed.After PCR amplification,the target fragment was cloned into prokaryotic expression vector pET-28a.The recombinants were transformed into the host bacteria BL21.After IPTG induction,the expression products were analyzed by SDS-PAGE.[Result] A 500 bp band with the expected size appeared in the RT-PCR products.After the pMD18-T-IL2 was identified by double digestion,an approximately 500 bp fragment was produced,which indicated successful cloning of the gene.After the pET-28a-IL2 was identified by restriction enzyme digestion and PCR,a 500 bp fragment was produced,which indicated successful construction of the expression vector.As revealed by the SDS-PAGE analysis,a protein band with molecular weight of about 20 kDa appeared.[Conclusion] The canine IL-2 gene was cloned and expressed.