Construction and Expression of Eukaryotic Expression Vector for M-CSF and EGFP Fusion Proteins

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 transfected NIH3T3 cells was observed under the fluorescence microscope 20 hours after transfection.RT-PCR analysis of M-CSF 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.