Therapeutic Effects Of Murine Ipr1 Recombinant Bcg On Mycobacterium Tuberculosis Infection In Murine Model

ObjectiveAt First, coding sequences of GFP, Orim and Ipr1 were cloned into pBudCE4.1 plasmid, which had multiple promoters to become the shuttle plasmid pBOGI. To construct the recombinant BCG with Ipr1 strain and to evaluate the effect of recombinant BCG on Mycobacterium tuberculosis infection of murine. This provides the groundwork for the exploring mechanism of the innate immunology against tuberculosis.Methods1. The coding sequence of Ipr1 was amplified from the total RNA of thymus of C57BL/6J mouse by RT-PCR, inserted into pEGFP-C1 to construct pEGFP-Ipr1.2. To Code sequences of GFP, Orim and Ipr1 were cloned into pBudCE4.1 plasmid, which had multiple promoters to become the shuttle plasmid pBOGI. A549 cells were transfected with the pBOGI by Lipo-fectAMINE2000, and the expression of the gene Ipr1 and EGFP would be observed by Fluorescence microscope,RT-PCR, immunohistochemistry, and Western Blotting.3. Constructing the recombinant BCG strain with Ipr1 that can targetedly deliver the shuttle coexpression plasmid pBGOI into macrophages to express the gene Ipr1, and to detect the expression of the recombinant BCG in vitro and in vivo.4. The mice BALB/c infected with Mycobacterium tuberculosis were intranasally treated with PBS, BCG, and the recombinant BCG, respectively. The mice were sacrificed after the final treatment. The numbers of viable bacteria in the lungs and the spleens were counted. The weight indexes of the lungs and the spleens were calculated. ELISA was used to detect the levels of IFN-γ,IL-10, and TNF-αin serum. The expression of Ipr1 in tissue was observed by immunohistochemistry. Lungs and spleens were prepared for pathological analysis.Results1. The Ipr1 gene from the thymus of C57BL/6J mouse were successfully obtained. The pEGFP-Ipr1 were successfully constructed.2. The pBOGI were constructed successfully, which were confirmed by DNA sequencing restriction and enzyme analysis. The expression in transfected A549 cells was successfully observed by RT-PCR, immunocytochemistry, Western Blotting, and so on.3. The recombinant BCG were successfully constructed. The expression of Ipr1 was detected by RT-PCR and immunohistochemistry in vitro and in vivo.4. The recombinant BCG group showed significantly reduce in number of colony forming units ,and the weight indexes of the lungs and spleens compared with the PBS group and the BCG group in the same murine strain. The recombinant BCG group had high level of IFN-γin serum. The expression of Ipr1 in lung and spleen was found in the recombinant BCG group. The pathological changes in lungs of the recombinant BCG group and the BCG group were localized, while those in the PBS group were extensive. There was no significant changes in the spleen.Conclusions 1. The coding sequences of GFP, Orim and Ipr1 would be coexpressed in murine macrophages.2. The recombinant BCG with Ipr1 gene was successfully constructed, and it gets excellently targeted delivery property and mainly distributes in the macrophages.3. The recombinant BCG with Ipr1 has therapeutic effects on murine Mycobacterium tuberculosis infection of murine.4. As a new therapeutic vaccine against Mtb, the recombinant BCG has great value of further exploration and application.