The Study Of The Impacts And Mechanisms Of Lentiviral Vector-mediated SiRNA Targeting TACO Gene On The Macrophages Rsisitance Against Mycobacterium Tuberculosis

Objective:To construct and screen for lentiviral interference vector,which can be specifically expressed TACO genes si RNA. To reseach and explore the effect and mechanism in the inhibition of macrophage intracellular bacteria. To provide basis and direction for subsequent reseach of targeted against Mtb infection in vivo. Method:1. Choosing three target sequence, According to TACO gene m RNA sequence, designing corresponding sh RNA in accordance with si RNA design principle and a negative contrast sh RNA sequences by random disturb. For each sh RNA each to design a pair of single oligodeoxynucleotide,the express double-stranded DNA is formed after low temperature annealing.Digesting the PSICOR lentiviral vector plasmid by restriction enzymes.Inserting annealed double-stranded DNA into the incision,getting the plasmid of PSICOR-TACO-si RNA. Confirmed correctly by enzyme digestion and sequencing identgification,determine its naming PSRT 1-4,the p SRT4 lentivirus expression plasmid is negative contrast sh RNA.2. Packaging virus particles by mixing the restructured lentiviral interference vector(PSRT1~4), the envelope protein plasmids p MD2.G and the lentiviral packaging plasmids ps PAX2,according to a certain percentage.The supernatant was collected containing lentiviral particles, concentrated virus particles after centrifugal ultrafiltration(Named LV- p SRT1-4, respectively),Then,infected 293 T cells by hole dilution method, via measuring and counting the number of the fluorescence expression cell, calculated virus drops degree.The packed virus particles infected RAW264.7 cell in suitable titer for 48 hours and 72 hours,Cracking cells extracted total RNA and total protein to detect macrophages TACO in expression in the level of gene transcription and translation with fluorescent quantitative PCR. and Western Blot protein imprinting method.Selected the PSRT1 as the most effectiveness restructured lentiviral interference vector.3. The LV-p SRT1 and the LV-PSRT4 packed virus particles infected RAW264.7 cells,After 72 hours, the cells were infected with the Mtb in a certain proportion for 4 hours,washed the bacteria wasn’t engulfed thoroughly and the time is 0h.The group of Mtb infection set time point 0 h, 24 h, 48 h and72 h.At various time points, destroied the cell membrane to release the bacterium wasn’t killed with 1%triton, respectively.Coated the plate evenly after dilution,counted the bacterial colony and statistical compared the differences between the groups.4. With the Alexa Fluor ® 647 dye dyeing the H37 Rv MTB to infect RAW264.7 cells which are the uninfected or by the LV- p SRT1 or LV- p SRT4 infected respectively, after 24 h, with Dy Light 405 sheep resistance against rabbit Ig G immunofluorescent labeling macrophages lysosomal.With the laser confocal microscopy observation group intracellular MTB and lysosome localization, to explore the influence of MTB phagosome and lysosome fusion rate when TACO expression has decreased.5. The LV-p SRT1 and the LV-PSRT4 packed virus particles infected macrophages cells,after 72 h,MTB H37 Rv infected macrophages cells according to the proportion of MOI is 5,at the same time,seting up the control group without MTB infection.After 24 h, cracking each cell, detecting the expression level of LC3 by the western-blot method and evaluating the effect on autophagy level when TACO expression decreased. Results:1. Enzyme digestion and sequencing results show that the TACO specific si RNA lentiviral interference vector PSRT1, PSRT2, PSRT3 and negative control PSRT4 constructed successfully.2. 90% of the cells can be detected fluorescence after lentiviral virus particles infected 293 T cells for 24 hours.The drop degree can reach more than 1~2×108 TU/ML, which has been completely conform to required transfection. Macrophages be infected with lentiviral virus particles for 48 hours,their fluorescence efficiency still can be as high as 90%.3. Fluorescence quantitative PCR results showed the TACO gene inhibition rate of LV-p SRT1, LV-p SRT2, LV-p SRT3 were 85.24%, 80.71%, 42.49%, the strongest inhibitory effect is PSRT1. Western Blot immunoblot results agree with the fluorescent quantitative PCR results, according to the gray scale scanning, the TACO relative expression of LV-p SRT1, LV-p SRT2, LV-p SRT3 were 0.31, 0.38, 0.47, the strongest inhibitory effect still is PSRT1.4. The burden of bacterium results showed the LV-PSRT1 could promotethe macrophage ability of killing the Mtb significantly,Compared with LV- p SRT4 infection group, the MTB surviving relative inhibition rate in macrophages in LV-p SRT1 infection group at 24 h, 48 h and 72 h were 47.26%, 53.00% and 47.26% respectively5. Compared with LV- p SRT4, LV- p SRT1 MTB infection can significantly improve the MTB and lysosome localization rate(PSRT1:75.67% vs Con group: 75.67%), prompted that downgrade TACO expression can significantly enhance the MTB phagosome and lysosome fusion rate.6. Western Blot immunoblot results showed LC3 expression and LC3 â…¡/LC3-â…  ratio has no significant effect on RAW264.7 cells in the LV- PSRT1,prompt that downgrade TACO expression had no obvious effect on macrophage autophagy level. Conclusion:1. Successfully constructed recombinant lentivirus vectors which sh RNA targeted TACO gene in mice, and packaging successfully obtained lentiviral vectors can express TACO gene specificity sh RNA.2. Selecting the TACO-sh RNA1(PSRT1) as the most effective interference sequences in the real-time fluorescent quantitative PCR and Western blot detection method, which laid a foundation for further verify its bacteriostasis function.3. Confirmed PSRT1 can significantly enhance the macrophage ability of killing MTB intracellular.4. Founded that the inhibition of TACO expression in macrophages can effectively promote the expression of MTB phagosome fusion with lysosomes, suggest the existence of the TACO may be one of the main mechanisms of hampering the MTB Phagosome mature in macrophage.5. The study shows thatinhibition of TACO expression in macrophages has not significantly effect on the level of autophagy, prompt that PSRT1 promoting the fusion of MTB phagosome and lysosome is not through autophagy- lysosome way, The mechanisms are still not well understood and need further study.