小鼠重组釉蛋白基因稳定表达细胞系的建立

Construction of the stable cell line expressing the mouse recombinant enamelin gene

目的:构建小鼠重组釉蛋白基因的真核表达系统,并建立稳定表达该蛋白的细胞系。方法:取初生小鼠牙胚组织,提取RNA,用RT-PCR技术扩增釉蛋白基因片段,经双酶切后克隆至真核表达载体pcDNA3.1TM/myc-His(-)B上,转化大肠杆菌E.coli DH5α,中提质粒,将该重组表达质粒转染至HEK 293A细胞,用G418筛选出阳性克隆,并检测釉蛋白的表达水平。结果:通过测序表明,小鼠釉蛋白基因被成功地连接到了真核表达载体上。将该表达系统转染HEK 293A细胞后,进行Western Blot检测,证明其中有相对分子质量约32000的釉蛋白表达。结论:成功构建了小鼠重组釉蛋白真核表达载体,建立了稳定细胞系,为获得高纯度的釉蛋白,为进一步研究蛋白质功能奠定了基础。

Objective： To construct a mouse recombinant enamelin eukaryocyte expression system, and establish the stable cell line which can produce the protein continuously. Methods： The mRNA transcript from the 3-day mouse jaw was extracted, and the enamelin gene fragment amplified with Rt-PCR techniques. Then the PCR product was cat with two restriction enzymes, and subcloned into the eukaryotic gene expression vector pcDNA3.1TM/myc-His （-） B. The recombinant plasmid was transformed into E. coli DH5ct bacterial cells, and harvested with plasmid midi kit. The recombinant expression plasmid was transferred to the HEK 293A eukaryocyte ceils, cultured selectively with 800 mg/L G418, and examined with SDS-PAGE and Western Blot at the protein level. Results： The mouse enamelin gene was cloned to the eukaryotic expression plasmid successfully by sequence measuring. After the recombinant plasmid was transferred into the HEK 293A cells, about 32 000 enamelin protein was checked out by SDS-PAGE and Western Blot. Conclusion： The recombinant eukaryocyte expression plasmid and the stable cell line were established. This is a basic research to obtain high-yeild biologically active enamelin protein, which may facilitate further investigation of its function.