作　　者： ; ; ; ; ;

机构地区： 华南农业大学动物科学学院

出　　处： 《华南农业大学学报》 2005年第1期102-105,共4页

摘　　要： 从经ConA活化的4周龄岭南黄鸡脾脏淋巴细胞中分离提取总RNA,经过反转录PCR(RT PCR)扩增出鸡白细胞介素 2(IL 2)cDNA片断.PCR产物克隆至pGEM TEasy载体,重组质粒命名为IL 2 T,经菌落PCR与限制酶切鉴定,然后测序,结果表明克隆片断长度为432bp,与预期大小一致,为鸡IL 2基因的编码区全长.将IL 2 T克隆重组质粒进行双酶切(ClaI与XbaI),回收IL 2片断插入同样酶切的毕赤酵母表达质粒pPICZα C,构建重组IL 2 C表达质粒,为鸡IL 2在毕赤酵母中的表达奠定基础. The Lingnanhuang chicken interleukin-2 (IL-2) cDNA fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from Con A stimulated,cultured spleen lymphocytes of four-week-old chicken. The RT-PCR product was inserted into the pGEM-T cloning vector. Restriction analysis and PCR were performed to identify the clone containing the DNA fragment of interest, and then the cDNA fragment was sequenced. The result showed that the length of obtained fragment was 432 bp and was the same as that of the chicken IL-2 gene open reading frame. The plasmid IL-2-T was digested with the enzymes Xba I and Cla I, the IL-2 fragment was recovered, and the IL-2 fragment was inserted into the plasmid pPICZα-C digested with the same enzymes. Also, restriction analysis and PCR were performed to identify the clone containing the plasmid IL-2-C, and then the cDNA fragment was sequenced. The result showed that the ORF was correct.These results provided foundation for the expression of IL-2 in yeast expression systerm.

关 键 词： 白细胞介素 酵母表达 克隆 表达质粒 重组质粒 基因 毕赤酵母 酶切 菌落

领　　域： [农业科学] [农业科学] [农业科学] [生物学]