绵羊白细胞介素2基因的克隆与序列分析

Cloning and Sequencing of Ovine Interleukin 2 Gene

为与腐蹄病节瘤拟杆菌纤毛蛋白基因构建融合基因表达载体 ,利用健康绵羊血扩增和克隆了设计有特殊酶切位点的绵羊白细胞介素 2 ( Ovi IL-2 )。从绵羊血中分离白细胞 ,用 TRIZOL试剂盒提取绵羊白细胞RNA,以 RNA为模板 ,用设计的 Ovi IL-2 3′引物进行 RT-PCR,扩增 Ovi IL-2 c DNA;以 Ovi IL-2 c DNA为模板 ,用 Ovi IL-2的 5′引物和 3′引物进行 PCR,扩增设计有 Bam H 和 Eco R 限制性酶切位点的 Ovi IL-2。经1 .5%琼脂糖凝胶电泳分析和 Xba 酶切鉴定 PCR产物后 ,将 Ovi IL-2 PCR产物与质粒 p Bluescript SK( + )连接 ,进行 Ovi IL-2基因克隆 ,在含 X-gal和 IPTG的 LB平板上 ,挑选白色单一菌落进行重组质粒筛选 ,用Bam H + Eco R 或 Eco R 酶切提取重组质粒 ,琼脂糖凝胶电泳出现 1条 50 0 bp大小的带 ;用 Eco R 或Eco R + Xba ,Bam H + Xba 酶切重组质粒分别出现 2 70 bp和 2 30 bp大小的带 ,从而筛选出重组质粒p Blue-Ovi IL-2。将筛选出的 p Blue-Ovi IL-2进行序列分析 ,证明克隆的 Ovi

To construct an expression plasmid harbouring fused ovine IL 2 and D.nodosus Pilin gene,the ovine IL 2 gene with synthesized primers with EcoRⅠ and BamHⅠ recognition sequence at 5′ and 3′ end respectively was amplified and cloned from the peripheral blood of ovine.Total ovine RNA was extracted from ovine blood by Reagent TRIZOL method.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 cloned into the pBluescript SK(+) vector.Then a recombinant plasmid named pBlue IL 2 was constructed.Sequence analysis confirmed that the amplified IL 2 gene is identical to the reported ovine IL 2 gene.