Immune responses induced by mucosal immunization with Mycobacterium tuberculosis EsxV subunit vaccine
BAI Lu1,2, NING Huan-huan2, KANG Jian2, LIANG Xuan3, XIE Yan-ling1,2, PENG Yu-jun2, ZHANG Jing-yao2, LU Yan-zhi2, BAI Yin-lan2
1. School of Life Sciences,Yan’an University,Yan’an 716000,China; 2. Department of Microbiology and Pathogen Biology, Basic Medical School, Air Force Medical University,Xi’an 710032,China; 3. College of Life Sciences,Northwest University,Xi’an 710069, China;
Abstract:This study investigated the immune responses induced by mucosal immunization with Mycobacterium tuberculosis antigen Rv3619c (EsxV) in mice. The esxV gene was amplified by PCR and cloned into the prokaryotic expression vector pET28a(+). Recombinant E. coli were induced to express the EsxV protein. Protein expression was identified by SDS-PAGE electrophoresis and western blotting. The recombinant EsxV protein was purified by affinity chromatography. BALB/c mice were immunized with EsxV and/or cyclic diadenosine monophosphate (c-di-AMP) adjuvant via the nasal mucosa. Specific antibody levels and subclasses were detected with ELISA. Splenocyte proliferation was detected with MTS assays. Cytokine expression levels in the spleen and lung were detected by qRT-PCR. Cytokines secreted by splenocytes were detected by ELISA. The prokaryotic expression vector pET28a (+)-esxV was successfully constructed. The recombinant EsxV protein was induced and expressed, then purified by affinity chromatography. EsxV induced significant humoral immune responses, but modest cellular immune responses, through mucosal immunization. Mucosal inoculation of EsxV with c-di-AMP induced an increase in specific IgG levels, EsxV protein-specific splenocyte proliferation, and spleen and lung IFN-γ transcription. c-di-AMP significantly enhanced EsxV-specific secretion of the IFN-γ, IL-2, IL-10 and IL-17 cytokines, but did not induce the expression of the inflammatory factors TNF-α and IL-6. The subunit vaccine constructed by EsxV and c-di-AMP induced specific humoral and cellular immune responses, which should be further used in the development of novel TB subunit vaccines.
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