鸡IL-18成熟蛋白基因的克隆及在大肠杆菌中的表达

Cloning and Expression of Chicken Mature IL-18 Gene in E.coli

目的 以鸡脾细胞mRNA为模板扩增编码鸡IL 18成熟蛋白的cDNA ,并在大肠杆菌中进行表达。方法 用PHA和LPS激活的AA肉鸡脾细胞 ,提取mRNA ,以RT PCR法扩增出编码鸡IL 18成熟蛋白的cDNA并测序 ,与pET 2 8b载体构建重组质粒 ,并在大肠杆菌BL2 1(DE3)plysS中表达 ,用SDS PAGE检测表达产物。结果 所克隆的核苷酸片段包含了鸡IL 18全部成熟蛋白编码基因 ,其大小为 5 0 7个核苷酸 ,编码 16 9个氨基酸 ;构建的重组表达载体在大肠杆菌BL2 1(DE3)plysS中表达出相对分子质量为 190 0 0的重组蛋白。结论 已成功克隆和表达了鸡IL 18成熟蛋白基因。

Objective To amplify the cDNA encoding chicken mature IL 18 protein from the mRNA of chicken splenocytes and express in E.coli. Methods Extract mRNA from the chicken splenocytes stimulated with phytohemagglutinin (PHA) and lipopolysaccharide (LPS), then amplify the cDNA encoding chicken mature IL 18 protein and sequence. Insert the cDNA into vector pET 28b and transform to E.coli BL21(DE3) plys S for expression. Identify the expressed product by SDS PAGE. Results The cloned cDNA fragment, consisting of 507 nucleotides encoding 169 amino acids, covered the whole chicken mature IL 18 gene. A recombinant protein with a relative molecular weight of 19000 was expressed in E.coli. Conclusion Chicken mature IL 18 gene was successfully cloned and expressed in vitro at first in China.