小鼠固醇载体蛋白2原核表达载体的构建与表达纯化  

Construction,expression and purification of a prokaryotic expression vector for sterol carrier protein 2

作　　者：胡水旺[1] 孙明晶[1] 夏高晓[1] 邓鹏[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理学教研室、广东省蛋白质组学重点实验室,广州510515

出　　处：《解放军医学杂志》2010年第10期1219-1221,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家重点基础研究发展(973)计划项目(2010CB529704);长江学者和创新团队发展计划(IRT0731);国家自然科学基金委员会-广东省人民政府自然科学联合基金重点项目(U0632004);国家自然科学基金项目(30670828);广东省科技计划项目(A1090202)

摘　　要：目的构建小鼠固醇载体蛋白2(SCP-2)融合蛋白表达载体,并在原核细胞内表达及纯化获得具有活性的融合蛋白,为进一步研究SCP-2的生物学功能提供基础。方法提取BALB/c小鼠肝脏组织总RNA,通过RT-PCR扩增得到鼠SCP-2编码序列,然后将该编码序列克隆到带有His标记的载体pET14b上,重组质粒经PCR、酶切及测序鉴定正确后转化大肠埃希菌BL21(DE3),用异丙基β-D硫代半乳糖(IPTG)诱导融合蛋白表达,用镍离子亲和层析的方法纯化融合蛋白并进行复性,然后切取分子量正确的条带用SDS-PAGE及质谱技术进行鉴定。结果重组质粒经PCR、酶切和测序鉴定证明载体构建正确。融合蛋白表达纯化后获得了分子量约59kD的融合蛋白,符合预期大小,并经质谱分析证明该融合蛋白的表达正确。结论成功构建了带His标签的SCP-2原核表达载体,并成功表达及纯化出该融合蛋白,为深入研究SCP-2的相关生物学功能提供了一个重要的工具。Objective To construct a prokaryotic expression vector expressing mouse sterol carrier protein Z （SCP-2）, and then transform the constructed plasmid, after expression and purification, into prokaryotic cells to obtain the active fusion protein, so as to provide a useful tool for the further study of the related biological functions of SCP-2. Methods The total RNA was extracted from the liver tissue of BALB/c mice, and the corresponding coding sequences of mouse SCP-2 （GenBank accession No. NM-011327） were amplified by RT PCR and then cloned into the His-tagged vector pET14b. The constructed vector was then transformed into E. coli BL21 （DE3）, which had been verified by PCR and double digestion with restriction endonuclease, followed by sequencing. After IPTG induction, the recombinant protein with His-tag was purified with Ni2＋ -NTA affinity chromatography method and then renatured. The purified protein was then identified by SIT, PAGE, finally, the protein band with the right molecular weight was picked and analyzed with MALDI-TOF/ TOF mass spectrometry technology. Results The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant plasmid was correctly constructed. The molecular weight of the purified fusion protein was 59KD by SDS- PAGE, which was identical to the molecular weight of mouse SCP-2 as predicted, and it was verified that the protein band was mouse SCP 2 with mass spectrometry analysis, Conclusion The prokaryotic expression vector with His-tag for SCP-2 fusion protein has been successfully constructed and effectively expressed, and the purified fusion protein has been verified, which can serve as a convenient and useful tool for the further study of the related biological functions of SCP-2.

关 键 词：固醇载体蛋白类 克隆 生物 基因表达 

分 类 号：R746.2[医药卫生—神经病学与精神病学]