摘要
目的构建人蔗糖酶(h SUC)基因原核表达载体,诱导表达获得融合蛋白。方法采用反转录PCR法扩增得到h SUC基因片段,并将其克隆到表达载体p ET-28a(+)中,用异丙基硫代半乳糖苷(IPTG)诱导在大肠杆菌中表达,采用Ni柱亲和层析纯化重组蛋白,对表达蛋白进行SDS-PAGE和Western blot鉴定。结果克隆得到1482 bp目的基因,酶切和测序证实h SUC基因片段成功插入到p ET-28a(+)表达载体。表达的融合蛋白质相对分子质量(Mr)为61 240,Western blot结果显示融合蛋白可以与h SUC抗体特异性结合。结论成功在大肠杆菌中表达和纯化h SUC融合蛋白。
Objective To construct prokaryotic expression vector p ET-28a（ ＋）-human sucrase（ h SUC） and express h SUC fusion protein in E. coli. Methods The h SUC gene fragment was amplified by reverse transciption PCR（ RT-PCR） and cloned into p ET-28a（ ＋） vector to construct the prokaryotic expression vector p ET-28a（ ＋）-h SUC. The recombinant plasmid was then transformed into E. coli BL21. Hisdidine（ His）-tagged fusion proteins were induced by isopropyl-beta-D-thiogalactopyranoside（ IPTG） and purified by nitrilotriacetic acid（ Ni-NTA） agarose resin. The purified fusion proteins were identified by SDS-PAGE and Western blotting. Results RT-PCR showed that sub-clone of h SUC was about 1482 bp. The recombinant plasmid was correctly constructed as demonstrated by sequencing and restriction enzyme analysis. The molecular mass of the fusion protein was about 61 240. Western blotting showed that the fusion proteins bound specifically to h SUC antibody.Conclusion The h SUC protein has been successfully expressed and purified in E. coli.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2015年第12期1629-1632,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
贵州省科技厅社会发展攻关项目[黔科合SY字(2012)3091]
2010年遵义医学院青年科研启动基金资助(F-501)
