Construction And Identification Of Taco Specific Small Interfering RNA Target Expression System

Suppressing the fusion of phagosome and lysosome is one of the main mechanisms for Mtb to survive in macrophage (Mφ). Recent researches demonstrated that TACO, an endogenous Tryptophan-aspartate containing coat protein of Mφ, plays a crucial role in the survival of Mtb within Mφ. TACO is recruited and localized to phagosomes by living, but not dead, Mtb. Through retaining TACO on the phagosome membrane, viable Mtb could prevent their delivery to lysome, thus evade the damage of immunity system. If the expression of TACO were interrupted, Mtb could be readily transported to lysosomes followed by their degradation. Since the primary intracellular niche of Mtb is Mcp, it may be an effective strategy to control the infection of Mtb by specially suppressing the expression of TACO in Mcp. To achieve the specific suppression of TACO expression in Mφ, some taco gene specific siRNA expression plasmid pSTl-pST4 were constructed and identified in this study, the capacity of pSTl-pST4 to suppress the expression of TACO in macrophage was detected. To achieve the targeted delivery of these plasmid, a recombinant M smegmatis harboring taco specific siRNA expressing plasmid was constructed and identified. We hope it can lay a theoretical foundation for opening a new thinking of Mtb control.Part I:Construction of the TACO special siRNA expression plasmidObjective:To construct the TACO special siRNA expression plasmid and build foundation for the following research.Method:Three pairs of complementary single DNA was designed according to the invitrogen on-line siRNA designing software and annealed to form the coding sequences of TACO special siRNA. A control siRNA expressing sequence which has the same base composition as siRNA1 but a scrambled sequence was also designed. A loop sequence was designed to link the lateral interfering sequence. Restriction ends of BamH I and Bbs I were designed at the end of the lateral interfering sequence for coloning purpose. The four double stranded siRNA coding sequences were cloned into pGPU6/GFP/Neo by BanHⅠand Bbs I to give pST1-4 respectively.Results:Restriction endonuclease assay and sequence analysis showed that all of the four siRNA coding sequence were identical to the expectant sequence and cloned into pGPU6/GFP/Neo.Conclusion:TACO special siRNA expression plasmids pST1,pST2,pST3 and a control siRNA expressing plasmid pST4 were constructed successfully.Part II:Identification of the expression characteristic and the effects of the tacospecific siRNA expression plasmid on suppressing of TACO expressionObjective:To identify the expression characteristic and the effects on suppressing TACO expression of taco specific pST1-4 in RAW264.7 and 293 T cells.Method:Taco gene was amplified from RAW264.7 cell line by RT-PCR and inserted into pQE30 to obtain the template of real time PCR for standard curve. Based on our previous study, three different methods were used to identify the effects of the taco specific siRNA expression on suppressing of TACO. (1) RAW264.7 cells were transfected with pST1-pST4 respectively, and the expression of TACO was detected at the mRNA level by real time RT-PCR and at the protein level by western blot. (2) RAW264.7 cells were transfected with pST1-4 respectively. After transfection, anti-TACO polyclonal antibody (1:300) was used as primary antibodies for the detection of TACO. The proteins were visualized with RFP labeled goat anti-rabbit antibody, intensity of red fluorescent between transgenic pST group and non-transgen ic group were detected to evaluate the effects of siRNA plasmid on the expression of taco. (3) Because having no endogenous taco expression and having high transfection efficiency, plasmid expressing the fusion protein of RFP and taco was constructed (pCDNA3.1-Red-taco). The 293T cells were cotransfected with pCDNA3.1-Red-taco and siRNA pST1,pST2,pST3,pST4, after transfection, the protein level was measured by western blot.Results:Because of low efficiency of transfection, we detected no distinction by real time RT-PCR or western blot in RAW264.7. However, the reduction was observed in cell immunity fluorescence of the RAW 264.7 after being transfected with pST interfereing plasmid at the single-cell level. Furthermore, pST interfering plasmid significantly down-regulated the expression of TACO pCDNA3.1-Red-taco transfeced 293T cell.Conclusion:pST interfering plasmid can significantly down-regulated the expression of TACO.PartⅢ:Construction and identification of recombinant Mycobacterium smegmatis harboring pSTMObjective:To construct a recombinant Mycobacterium smegmatis harboring pSTM, achieve the target delivery of these plasmids.Method:ORIM, the replicon of Mycobacterium was cloned into siRNA expression plasmid pST1-4 to give pSTMl-4, The recombinant pSTMl-4 were electroporated into Msl-2c to give rMs-pSTMl-4, the target performance of rMs-pSTMl-4 was evaluated by acid-fast stains and fluorescence assay.Results:rMs-pSTMl-4 were constructed successfully. Acid-fast stains and fluorescence assay showed that rMs-pSTM can target to macrophage.Conclusion:rMs-pSTM which can target to Macrophage were constructed successfully.