Cloning and expression of murine Desmin gene and its bioinformatics analysis

【Objective】 To construct an eukaryotic/prokaryotic expression vector for murine type III intermediate filamentary Desmin protein，to clarify the biological functions of murine Desmin protein，and to provide a theoretical basis for identifying Desmin-interacting proteins and exploring their action networks in in vitro and intracellular expression systems.【Method】The Desmin gene was cloned by RT-PCR，and the prokaryotic expression vector pGEX-4T-1-Desmin^-Flag and the eukaryotic expression vector pcDNA3.0-Desmin^-Flag were constructed respectively，and the expression of the prokaryotic expression vector was induced by IPTG. ProtParam，ProtScale，TMHMM-2.0，SignalP-5.0，SOPMA and SWISS-MODEL were used for bioinformatics analysis of Desmin protein.【Result】 The murine Desmin gene was 1410 bp long. pGEX-4T-1 vector and pcDNA3.0 vector were used to successfully construct the prokaryotic expression vector pGEX-4T-1-Desmin^-Flag and the eukaryotic expression vector pcDNA3.0-Desmin^-Flag respectively. Prokaryotic expression vector pGEX-4T-1-Desmin^-Flag was transformed into Escherichia coli and successfully expressed a 70 kD fusion protein Desmin^-Flag， after induction by IPTG. The eukaryotic expression vector pcDNA3.0-Desmin^-Flag was transfected with BHK-21 cells and the Flag tag was detected at 56 kD by Western blotting，in other words， the Desmin protein was successfully expressed in eukaryotic cells and was mainly localized in the cytoplasm. Desmin protein consisted of 470 amino acids residues with a molecular weight of 54 kD，a molecular formula of C₂₂₉₉H₃₇₂₂N₆₈₈O₇₅₅S₁₃ and a theoretical isoelectric point （pI）of 5.21，which was an unstable protein；its lipolysis coefficient was 79.94 and its mean hydrophilicity coefficient （GRAVY）was-0.721. It was presumed to be a hydrophilic protein；there was no transmembrane structural domain and signal peptide；its secondary structure consisted of α-helix（67.59%），random coil（22.39%），β-turn（1.92%）and extended chain（8.10%）.【Conclusion】 In the prokaryotic expression system，murine Desmin protein is mainly expressed in the form of inclusion bodies，while in eukaryotic cells，it is mainly expressed in the cytoplasm，with skeletal structure distribution，unstable physical and chemical properties，and no transmembrane domain or signal peptide. It is a hydrophilic protein，Desmin，as an important type III intermediate filament protein，plays an important role in signal transduction and interaction with related proteins in neuromuscular tissue.