摘要
目的构建含有LIGHT胞外段的原核表达载体,并在大肠杆菌中诱导表达。方法从由小鼠骨髓来源的未成熟树突状细胞中提取总RNA,经RT-PCR扩增LIGHT胞外段,克隆至原核表达载体pET-24a(+)中,构建重组表达质粒pET24-LIGHTECD。重组质粒经酶切鉴定及序列测定后,转化E.coli BL21,IPTG诱导表达,并用SDS-PAGE和Western blot进行检测。结果RT-PCR扩增出了525bp LIGHT胞外段的cDNA,经测序证实其序列正确。SDS-PAGE和Western blot分析证实重组质粒可表达出相对分子质量为20000的LIGHT胞外段蛋白。结论成功地克隆了小鼠LIGHT胞外段基因并在大肠杆菌中进行了表达,为进一步研究LIGHT的功能打下了基础。
Objective To construct a prokaryotie expression vector for the extraeellular domain of LIGHT （ LIGHTECD ） and express it in E. coll. Methods Total RNA was extracted from murine immature bone marrow-derived DCs and the cDNA of extracellular domain of LIGHT was amplified by RT-PCR. The recombinant plasmid pET24-LIGHTECD was constructed by cloning the extracellular domain of LIGHT cDNA into the prokaryotic expression vector pET-24a（ ＋ ）. After the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing, the pET24-LIGHTECD was transformed into E. coli BL21 and induced with IPTG. The expressed protein was analyzed by SDS-PAGE and Westem blotting. Results A 525 bp of LIGHT extracellular domain cDNA was amplified by RT-PCR and the sequence was confirmed by DNA sequencing. SDS-PAGE and Westem blot analysis showed that a protein with molecular weight of 20 000 was expressed in E. coli BL21. Conclusion The extracellular domain of LIGHT is cloned and expressed in E. coli successfully, which lays a basis for the further functional research of LIGHT.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2008年第4期389-391,399,共4页
Immunological Journal
基金
上海市青年科技启明星计划资助项目(06QA14017)
作者简介
张亚丽（1982-），女，甘肃兰州市人，硕士生，主要从事分子免疫学研究。（Tel）021—62233574；（E-mail）zhangyali002@hotmail．com.
通讯作者：江文正，（Tel）020—62233574；（E-mail）wzjiang@bio．eenu.edu．cn
