摘要
目的:构建人CIDE-3基因的原核表达载体,并在E.coliBL21(DE3)中表达。方法:提取人肝母细胞瘤细胞系HepG2细胞的总RNA,经RT-PCR扩增人CIDE-3基因片段,并将其克隆入原核表达载体pET28a(+)中,构建重组质粒pET28a(+)-CIDE-3。经限制性内切酶BamHI、XhoI双酶切鉴定及序列测定后,转化E.coliBL21(DE3),经IPTG诱导表达组氨酸融合蛋白。结果:获得全长为516bp的人CIDE-3基因片段。以构建的重组质粒pET28a(+)-CIDE-3转化E.coliBL21(DE3)后,经IPTG诱导,表达出相对分子质量(Mr)约为23000的重组CIDE-3蛋白。SDS-PAGE分析显示,表达的蛋白主要以不溶性包涵体的形式存在于E.coliBL21(DE3)的胞质中,表达量约占全菌蛋白的32%。结论:成功地构建了原核表达载体pET28a(+)-CIDE-3,并表达出重组CIDE-3蛋白,为进一步多克隆抗体的制备和凋亡的相关研究奠定了实验基础。
AIM： To construct a prokaryotic expression vector for human CIDE-3 gene, and to express the gene in E. coli BL21 （ DE3 ）. METHODS： The total RNA was extracted from human hepatocellular carcinoma cell line HepG2. CIDE-3 gene fragment was amplified by RT-PCR and was cloned into the pET28a（ ＋ ） vector. The recombinant vector was identified by restriction endonuclease digestion analysis and DNA sequencing, and then it was transformed into E. coil BL 21 （ DE3 ） through IPTG induction to express the target protein bearing His tag. RESULTS： A .516 bp of CI- DE-3 gene fragment was obtained. After E. coli BL21 （DE3） was transformed with recombinant vector pET28a （ ＋ ）-CI- DE-3 and through IPTG induction, the recombinant protein with relative molecular masse about 23 000 was obtained. SDS-PAGE analysis showed that the expressed product was mainly inclusion bodies, accounting for 32% of the total bacterial proteins. CONCLUSION： Recombinant expression vector pET28a（ ＋ ）-CIDE-3 is constructed successfully. The expressed CIDE-3 protein will be helpful to our further research.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第2期202-204,共3页
Chinese Journal of Cellular and Molecular Immunology
