Establishment and phenotypic characterization of an immortalized goat alveolar macrophage line

Abstract:

[Background] Although a variety of human and murine cell lines have been utilized as models for Brucella infection in vitro, there remains a lack of large animal cell models that can accurately simulate the infection process. [Objective] To obtain a stable passable goat alveolar macrophage line with single cloning characteristics and optimize the goat cell infection model of Brucella infection. [Methods] Goat alveolar macrophages were isolated by perfusing goat lungs with PBS under sterile conditions. An immortalized goat alveolar macrophage cell line was established by transfection with SV40 large T antigen (SV40LT) and puromycin resistance screening. The characteristic surface marker of mononuclear macrophages, CD₁₄, was detected by immunofluorescence and flow cytometry. The phagocytic ability of the established cell line was assessed with chicken red blood cells. Subsequently, the cell line was infected with Brucella vaccine strain M5, and the intracellular bacteria were counted at different time points. [Results] The alveolar macrophage cell line of goat was successfully isolated and constructed, and it had typical macrophage morphological characteristics, showing an irregular shape, pseudopodium protruding, large cells, and strong phagocytosis. After 10 d of cultivation, 95.40% of the primary cells specifically expressed CD₁₄, and 92.4% of the cells specifically expressed CD₁₄ after immortalization. Compared with the other three cell lines, the immortalized alveolar macrophage cells were sensitive to Brucella, with the sensitivity similar to that of RAW264.7 cells and higher than that of sheep testicular interstitial cell (STIC). [Conclusion] An immortalized goat alveolar macrophage cell line was successfully established, providing an in vitro experimental object for studying the pathogenicity and immune escape mechanism of Brucella.

Key words:SV40LT;lentivirus;Brucella;cell challenge model;flow cytometry