绵羊白细胞介素2基因的克隆与表达载体构建

Cloning and Construction Expressing Vector of Ovine Interleukin-2 Gene

从绵羊血中分离白细胞,提取绵羊白细胞RNA,利用RT-PCR、PCR扩增绵羊白细胞介素2(OvineIL-2)基因。将OvineIL-2PCR产物与TE载体定向连接,用SaLI和BamHI双酶切重组TE,利用低溶点胶回收500bp大小的片段;将其补平后与PPSD质粒连接,用EcoRI酶切鉴定,找出正向连接重组质粒。用BamHI酶切重组PPSD质粒,低熔点胶回收2500bp大小的片段;将其与经BamHI酶切去磷酸化的PME290表达质粒连接,筛选出阳性重组PME290,即为OvineIL-2基因表达载体。

IL2 is known to be as an adjuvant in many areas. The ovine IL2 gene with synthesized recognition sequence at 5` and 3` end respectivecy was amplified and cloned from the peripheral blood of ovine. Ovine IL2 gene was amplified with ovine RNA as template by RTPCR and PCR. The amplified fragment was subjected to restriction digest and coined into the TE and PPSD vector. An expression plasmid was constructed by cloning the IL2 gene into PME290. The PME290SDIL2 was transformed into the host competent cell PAK/2pfs and the recombinant IL2 was expressed in the supernatant of the cultures of the transformant cell PAK/2pfs.