摘要
应用RT-PCR技术从罗曼鸡脾淋巴细胞RNA中扩增鸡白细胞介素18(ChIL-18)全基因,并克隆和测序。结果表明,获得了ChIL-18全序列,其大小为597 bp。将其成熟蛋白基因(510 bp)亚克隆到原核表达载体pGEX-6P-1中,转化大肠杆菌BL21(DE3),在IPTG的诱导下表达融合蛋白(GST-ChIL-18)。SDS-PAGE可检测到分子质量为约46 kDa的融合蛋白,Western blot证实该融合蛋白可与鼠抗鸡IL-18单克隆抗体发生特异性反应。用MTT法测定表明,重组蛋白能明显促进鸡T细胞转化的活性。
Chicken interleukin-18 （ChIL-18） gene was amplified from total RNA extracted from Lohmann chicken splenocyte by reverse transcription-polymerase chain reaction （RT-PCR）. PCR products were cloned and sequenced. The results showed that the full-length of chicken IL-18 gene was 597 bp. A prokaryotic expression plasmid of chicken IL-18, pGEX-ChIL-18, was ob- tained by subcloning the encoding region of the ChIL-18 mature peptide into pGEX-6P-1. The recombinant ChIL-18 （rChIL-18） was expressed efficiently in pGEX-ChIL-18-transformed E. coli BL21 （DE3） induced by IPTG. The fusion protein was identified by SDS-PAGE and Western blot. The results showed that fusion protein GST-ChIL-18 was about 46 kDa and could react with monoclonal antibody for mouse anti-chicken IL-18. The recombinant protein could significant promote the transformed activity of chicken T-cell by methyl thiazolyl tetrazolium （MTT）.
出处
《四川农业大学学报》
CSCD
北大核心
2009年第2期218-222,共5页
Journal of Sichuan Agricultural University
基金
国家"十一五"科技支撑计划专项(2006BAD06A08)
关键词
鸡IL-18
克隆
原核表达
活性检测
chicken IL--18
eloning
prokaryotic expression
activity detection
