The Construction Of IFN-γ Promoter Luciferase Reporter System And Its Application In Screening Of Mycobacterium Tuberculosis Protein Regulated Expression Of IFN-γ

Tuberculosis, is a chronic infections disease caused by Mycobacterium tuberculosis. Tuberculosis has become one of the world’s most seriously public health problem. According to WHO estimates, there are around1/3of people infected with Mycobacterium tuberculosis in the world. There are8.6million new cases and1.3million deaths in2012. Mycobacterium tuberculosis infection induces a variety of cytokines production. IFN-γ is one of the most important cytokines which can resistant Mycobacterium tuberculosis infection in these cytokins. IFN-y is a Type II interferon produced primarily by activated T cells, NK cells, and it can activate macrophages which bear important significanc in clearing Mycobacterium tuberculosis. The aim of this study was to screen Mycobacterium tuberculosis protein which regulate IFN-γ production and further probe its regulatory mechnism.1. Selection of the candidate proteinWe concentrate on the lipoprotein, membrane proteins and secreted proteins of Mycobacterium tuberculosis H37Rv. We choose173proteins by bioinformatics and proteomics analysis, which were predicted to regulate the activity of IFN-γ. We designed PCR primers which carried the same restriction sites as soon as possible accoding to gene sequence of protein.2. Constructing the eukaryotic expression plasmid of candidate proteinIn this experiment, we utilized the Mycobacterium tuberculosis genomic DNA as template for PCR amplification. Then target gene was cloned to pcDNA3.1-V5/HisB vector.We successfully constructed eukaryotic expression plasmids altogether173. The experiment needed to build a lot of eukaryotic expression plasmid, so we took advantage of some simple experiments approach. First of all, we amplified the gene bearing the same restriction sites at the same time, and then digested the PCR product. Secondly, we measured the concentration of them and linked them with vector. And then transformed the linked production and picked a certain number of single bacterial colony shaking bacterium, extracting plasmids, identifying the plasmids with restriction endonuclease and sequencing. Finally, we made sequence analysis and screen successfully constructed plasmid. 3.Screening protein of Mycobacterium tuberculosis that regulate IFN-γ activitySince dual luciferase reporter gene system were selected for screening, therefore we needed to build IFN-γ promoter luciferase reporter plasmid. After predicting the sequence of IFN-γ promoter, we designed the primers of IFN-γ promoter. In this study, we extracted Hela cell’s genomic as template and construct pGL3-Basic luciferase reporter plasmid of IFN-γ promoter.Next eukaryotic expression plasmids and IFN-γ promoter luciferase reporter plasmid were cotransfected to Hela cell. Dual luciferase was assayed after treated36h. Protein Rv0720inhibited IFN-γ promoter, in dose dependent manner. Protein Rv3623activied IFN-γ promoter, in dose dependent manner.4. Researching the target protein in protein levelsFirst of all, we choose protein Rv3623and Rv072as research target. Protein Rv3623, Rv3763and Rv0720are belong to membrane phospholipoprotein. At the same time, protein Rv3763was reported to induce cell apoptosis. Although Protein Rv3763regulate IFN-γ promoter weakly in dual luciferase reporter gene system testing, we wanted to study the protein Rv3623, Rv3763and Rv0720regulated function to IFN-γ in protein level.Therefor their gene was cloned into PGX-KG vector and transformed into E.coli BL21. After the cell was induced by IPTG, we extracted inclusion bodies and deal with protein for endotoxin removal. Finally we treated RAW264.7cells with the protein and detected IFN-γ transcriptional level by real-time PCR and protein level by ELISA. Results suggest their ability to regulate IFN-γ was undected.