Effects Of AG85A On The Activity And Function Of Human Monocytes

Objective: To investigate the characteristics of the human monocytes transfected with the recombinant plasmid pcDNA3.1-Ag85A, and to study the expression of Ag85A gene and protein in human monocytes, in order to gain the immunocompetent monocytes. Methods: Centrifugalized and collected the monocytes from human peripheral blood mononuclear cells (PBMC). After primary culture in vitro, the purity of the monocytes was measured by flow cytometry The monocytes were transfected with the recombinant plasmid pcDNA3.1-Ag85A. The expression of the Ag85A gene in the monocytes was detected by RT-PCR. The synthesis of the antigen85 protein in monocytes was detected by Western Blot. The T lymphocytes were isolated from PBMC, and were co-incubated with the monocytes transfected with the recombinant plasmids. At the same time, set the pcDNA3.1 tansfected group, and the recombinant plasmid plus monocytes co-incubated group as the negative controls. After 3 day's incubation, detected the proliferation of the T cells by MTT method. Results: After 6 days'culture in vitro, a large number of adherent monocytes were acquired, highly expressed CD14, at about 98.35%. RT-PCR demonstrated that the gene Ag85A was transcribed well in the transfected monocytes, as well as the Western Blot confirmed that the protein antigen 85A was synthesized by the transfected monocytes. After co-incubation with the T lymphocytes, the monocytes transfected with the recombinant plasmids had the stronger capability of promoting the proliferation of T lymphocyte, compared to the negative controls. Conclusion: The Ag85A gene and protein could be expressed by the human monocytes through gene transfection. And the monocytes transfected by the recombinant gene had the capability of promoting the proliferation of T lymphocyte, which might offer a new way for the development of the vaccine and therapy of the patient who suffered both tuberculosis and AIDS.