Construction And Its Immune Response Of Ipr1/PPE68DNA Vaccine

Objective:To construct a new shuttle plasmid pBud-Iprl-PPE68-OriM, observe the expression of intracellular pathogen resistance1(Iprl) protein and PPE68antigen in the macrophage RAW264.7, and study the immune response induced by the Iprl/PPE68DNA vaccine in BALB/c mice.Methods:1. Iprl gene and the coding sequences of PPE68were cloned into the MCS sites of the plasmid pBudCE4.1, and the plasmid pBud68-Iprl was constructed. After identified by restriction endonuclease digestion and DNA sequencing, the plasmid pBud68-Iprl was transfected into the RAW264.7. The expression of Iprl and PPE68protein were detected by RT-PCR and Western-Blot. The coding sequences of Mycobacterium tuberculosis origin of replication (OriM) were cloned into the Nhe I site of the plasmid pBud68-Iprl, the shuttle plasmid pBud-Iprl-PPE68-OriM was constructed.2. The BALB/c mice were immunized with the shuttle plasmid pBud-Ipr1-PPE68-OriM. Two weeks after the last immunization, the levels of1gG2a, IL-12and IFN-y in serum were analyzed by ELISA. Specific proliferation of spleen lymphocytes and the quantity of CD4+and CD8+T cells were detected with MTT assay and FCM respectively. The tissue slices of lungs and spleens were observed at the same time.Results:1. The result of restriction endonuclease digestion and DNA sequencing showed that the shuttle plasmid Iprl/PPE68was constructed successfully. Iprl and PPE68protein were detected by RT-PCR and Western-Blot.2. The levels oflgG2a, IL-12and IFN-y and the quantity of CD4+and CD8+T cells were significant increase. The proliferative responses of spleen lymphocyte were not different from BCG group. The pathology analysis showed no obviously changes in lungs and spleens.Conclusions:1. The shuttle plasmid pBud-Iprl-PPE68-OriM was constructed successful, and Iprl and PPE68protein were expressed in the transfected macrophage RAW264.7.2. The Thl immune response was effectively induced by the shuttle plasmid pBud-Iprl-PPE68-OriM in BALB/c mice. This study lays a foundation for the future late-stage study about immune protection efficacy of the Iprl/PPE68DNA vaccine.