猪白细胞介素18成熟蛋白基因的克隆及在大肠杆菌中的表达  

Cloning and Expression of Porcine Interleukin-18 Mature Protein Gene in Escherichia coli

作　　者：郑敏[1] 金宁一[1] 张洪勇[1] 李昌[1] 尹革芬[1] 

机构地区：[1]解放军军需大学军事兽医研究所,吉林长春130062

出　　处：《中国兽医学报》2003年第5期430-432,共3页Chinese Journal of Veterinary Science

基　　金：国家 8 63计划资助项目 (2 0 0 1AA2 13 0 71)

摘　　要：通过 RT- PCR方法从用 PHA和 L PS刺激的猪脾脏细胞中扩增出猪白细胞介素 18(p IL - 18)成熟蛋白基因的c DNA,克隆到 T载体 p UCm- T后 ,序列测定表明 ,p IL- 18成熟蛋白基因核苷酸长度为 4 74 bp,编码 15 7个氨基酸。将该基因片段克隆到大肠杆菌表达载体 p ET- 2 8a构建重组质粒 p ETIL 18m,转化大肠杆菌 BL 2 1(DE3) ,并用 IPTG诱导。重组菌菌体裂解物 SDS- PAGE可检测到相对分子质量为 190 0 0的重组蛋白 ,免疫印迹法证实该重组蛋白可以与人 IL-Porcine interleukin 18 mature protein gene was amplified from porcine spleen cells stimulated with phytohemagglutinin(PHA) and lipopolysaccharide(LPS). PCR product was cloned into the T vector pUCm T for sequencing. The result showed the nucleotide sequence of this gene was 474 bp. Then, this pIL 18 mature protein gene was subcloned into the prokaryotic expressing plasmid vector pET 28a and transformed into host E.coli strain BL21(DE3) for expression. The expression of pIL 18 mature protein gene was identified by SDS PAGE and Western blotting. The results revealed it had a molecular weight of 19 000, and could be specifically recognized by the mouse monoantibody to human IL 18.

关 键 词：猪白细胞介素18 成熟蛋白 基因克隆 原核表达 RT-PCR 大肠杆菌 免疫佐剂 免疫调节剂 

分 类 号：R392.11[医药卫生—免疫学] Q78[医药卫生—基础医学]