摘要
目的构建小鼠24p3基因真核表达载体并观察其稳定转染的NIH3T3细胞中24p3/SIP24蛋白的表达情况,初步鉴定24p3/SIP24蛋白介导铁向细胞内转运的生物学功能。方法RT-PCR自小鼠肺组织中克隆24p3 cDNA。用基因重组技术构建24p3-pcDNA3.0真核表达载体并行酶切和测序鉴定。脂质体DOTAP方法转染NIH3T3细胞,48 h后用G418筛选阳性克隆并培养扩增至21 d。对阳性克隆的培养基上清中24p3/SIP24蛋白的表达用ELISA和W esternb lot分析,进一步用原子吸收分光光谱观察其介导铁向NIH3T3细胞内转运的生物学功能。结果成功克隆24p3 cDNA并构建24p3-pcDNA3.0真核表达载体,筛选出稳定表达24p3/SIP24蛋白的NIH3T3细胞株,24p3/SIP24蛋白在NIH3T3细胞上清中获得表达,并能介导铁向NIH3T3细胞内转运。结论24p3基因在NIH3T3细胞中获得表达,有良好的生物学活性,为进一步研究24p3/SIP24蛋白介导铁转运的生物学功能及作用机制奠定了基础。
Objective To construct the eukaryotic expression vector of mouse 24p3 gene and express it in NIH3T3 fibroblast cell line and verify its biological function of delivering iron into cells. Methods 24p3cDNA was cloned by RT-PCR and the eukaryotic expression vector of mouse 24p3 gene was constructed by gene recombination technique and the recombinant plasmid was verified by restriction enzyme digestion analysis and sequenceing, then transfected into NIH3T3 cells using DOTAP transfection reagent. Then the positive clones were selected and amplified. The expression of 24p3 gene in the culture supernatant of positive clones was analyzed by ELISA and Western blotting and its biological activity of delivering iron into cells was tested by atomic absorption spectrometer. Results The eukaryotic expression vector was constructed successfully. The NIH3T3 cells that stably expressed 24p3 were screened out. 24p3/SIP24 protein could express in the culture supernatant of positive clones and deliver iron into NIH3T3 cells. Conclusion The 24p3 gene was expressed successfully in the transfected NIH3T3 cells with good biological activity, which laid the foundation for further studying its biological function and mechanism involved in the iron metabolism.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2005年第17期1725-1728,共4页
Journal of Third Military Medical University
