摘要
目的构建真核表达重组体pEGFP-M-CSF,并鉴定其在小鼠成纤维细胞NIH3T3细胞能否正确表达。方法PCR扩增M-CSF基因,定向克隆入带有EGFP报告基因的真核表达载体,构建真核表达重组体pEGFP-M-CSF,脂质体介导将pEGFP-M-CSF转染入NIH3T3细胞,以一步法RT-PCR检测其在NIH3T3细胞中的表达情况。结果双酶切及测序鉴定证明成功构建真核表达重组体pEGFP-M-CSF,并在NIH3T3细胞中成功地表达了融合荧光蛋白,RT-PCR检测结果显示RT-PCR扩增产物与M-CSF目的基因片段大小相符,确实源于重组质粒转录后的mR-NA。结论真核表达重组体pEGFP-M-CSF的成功构建及在体外真核细胞中有效表达,为进一步研究M-CSF在真核细胞中的信号调控奠定了一定的实验基础。
Objective To construct the recombinant plasmid of eukaryotic expression vector containing M-CSF gene and transfect it into the NIH3T3 cells to study whether resulting in the expression of M- CSF and EGFP fusion proteins. Methods M-CSF gene was amplified by polymerase chain reaction （PCR）, and subcloned into an expression vector pEGFP-C1 containing the report gene of EGFP. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid pEGFP- M- CSF was transfected into NIH3T3 cells by using the liposome technique. M- CSF mRNA in NIH3T3 cell was analyzed by RT- PCR. Results The recombinant vector was identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. Bright green fluorescence in the cytoplasm and nucleus of the trans- fected NIH3T3 cells was observed under the fluorescence microscope 20 hours after transfection. RT - PCR analysis of M-C, SF mRNA in NIH3T3 cell after transfection demonstrated that the RT- PCR products were derived really from mRNA transcripted from the recombinant plasmid. Conclusion The fusion proteins of M- CSF and EGFP were successfully expressed in NIH3T3 cells. The results suggest that mammalian cells could express cytokine fusion proteins, which laid the foundation for further investigation into the biological activities of M-CSF.
出处
《实用预防医学》
CAS
2005年第6期1257-1260,共4页
Practical Preventive Medicine
基金
国家自然科学基金(30270684)
湖南省杰出中青年专家专项基金(02JJYB004)
