利用一对自行设计的引物,通过反转录-聚合酶链反应(RT-PCR),从ConA诱导的5周龄SPF鸡脾细胞中扩增出鸡白细胞介素2(IL-2)基因片段,其长度为439 bp,经酶切鉴定,初步确定为鸡IL-2基因.再将该IL-2基因片段定向插入真核表达质粒pcDNA3中,经DNA序列鉴定,表明构建的重组质粒含有IL-2基因,该基因的cDNA序列与Sundick报道的结果基本一致.然后,将IL-2基因片段插入原核表达载体pGEX-4T-1,并导入菌株BL21, 经诱导培养、蛋白提取和SDS-PAGE,获得分子量约为42 000的蛋白条带,表明所克隆的鸡IL-2基因在原核细胞中得到表达.
In this study,a cDNA fragment of chicken IL-2 about 439 bp was cloned from ConA- stimulated chicken spleen cells by reverse transcription (RT)-PCR with a pair of designed primers. Then it was inserted into pcDNA3, an eukaryotic expression plasmid by a directional link, identified by endonuclease and DNA sequencing, which conformed to chicken IL-2 gene fragment reported in the literature. This fragment was further inserted into a prokaryotic expressive plasmid pGEX-4T-1, and then it was transferred into the strain BL21. After the bacteria were induced and cultured, expressed protein was separated. A band with 42 000 of molecular weight was observed on SDS-PAGE. All these results suggest that the cloned gene of chicken IL-2 is expressed in prokaryotic cells.