Abstract: Objective To construct a recombinant lentiviral vector containing rat paralemmin-3 (PALM3) gene and detect its expression in alveolar macrophages (NR8383 cells). Methods Rat PALM3 gene fragments were amplified and obtained by polymerase chain reaction (PCR), and were cloned to pCV186-Cherry. After identification of pCV186-CherryPALM3 with PCR and DNA sequencing, the recombinant vector, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T packaging cells to generate recombinant lentiviral vector lenti-CV186-Cherry-PALM3. The titer of lenti-CV186-Cherry-PALM3 was determined by fluorescence labeling method. NR8383 cells were transfected with different concentrations of lenti-CV186-Cherry-PALM3 to obtain the optimal MOI according to transfection efficiency. The protein expression of PALM3 was detected by Western blot assay following transfection with lenti-CV186-Cherry-PALM3 under the optimal MOI. Results Rat PALM3 gene fragments were obtained by PCR and ligated into the pCV186 plasmid vector.The PCR and DNA sequencing results demonstrated that the recombinant plasmid pCV186-Cherry-PALM3 was successfully constructed. The recombinant lentiviral vector was successfully packaged, and its titer was 3.0×108 TU/mL. The optimal MOI value for the NR8383 cells was 20. The result of Western blot assay showed that PALM3 protein expression was significantly enhanced after transfection with lenti-CV186-Cherry-PALM3 (P＜0.05). Conclusion The recombinant lentiviral vector lenti-CV186-Cherry-PALM3 has been constructed, and efficiently expressed in alveolar macrophages,which offers a basis for further research of the effect of PALM3 on macrophage polarization.

Key words: macrophages, alveolar, paralemmin-3, recombinant lentiviral vectors, acute lung injury, acute respiratory distress syndrome