Construction And Identification Of MTOR Specific Small Interfering RNA Lentiviral Expression Vector

Objective:To construct and screen for expression of mTOR gene special siRNA lentivirusand using macrophage-specific promoter joint into recombinase-based lentiviralsiRNA expression vector, which can be specifically expressed in macrophages, toexplore the effect and mechanism in battling against mycobacterium tuberculosis invivo and vitro studies.Method:1. To choose three target sequences based on mTOR gene mRNA sequence,according to each target sequence by siRNA design principles to design appropriateshRNA, while one of shRNA sequences designed according to the length and basecomposition with a completely consistent, but the nucleotide sequence is randomscrambled sequence as a control shRNA sequences. For each shRNA each to design asingle-stranded oligodeoxynucleotide, To design and synthesis of four single-stranded oligodeoxynucleotide according to mTOR gene, slow annealing to formdouble-stranded by DNA, ligated into pSicoR vector plasmid, which was doubledigested by Hpa I and Xho I. The pSicoR-mTOR interference plasmid wereconstructed and named pSM series vector. After transformation of E. coli DH5α,positive clones were correctly identified after digestion, then DNA sequencing.2. Lentiviral expression vector pSM, lentiviral packaging plasmids psPAX2,envelope protein plasmids pMD2.G according to a certain percentage of transfected293T cells, the supernatant was collected containing lentiviral particles, aftercentrifugal ultrafiltration, concentrated virus particles. The293T cells were infectedwith lentiviral particles using fluorescence assay by dilution holes in the expressionof an inverted fluorescence microscope, and calculate the viral titer, suitable titer oflentiviral particles infected mice macrophage RAW264.7, after48h,observe a slowvirus particles RAW264.7cells. Using real-time quantitative PCR and Western blotdetect mTOR gene expression in macrophages. 3. The sequence of Cre were amplified by PCR from Cre-Amp plasmid and theninserted into Hind III/Xba I sites of plasmid pBudCE4.1-SP146to form therecombinant plasmid pBudCE4.1-SP146-Cre. After restriction endonucleaseevacuation correct,the recombinant plasmids above were sequenced.4. The sequence of SP146-Cre were amplified by In-Fusion cloning technologyfrom pBudCE4.1-SP146-Cre plasmid and then fused into pSico to form therecombinant plasmid pSico-SP146-Cre, The recombinant plasmids above weresequenced. The SP146promoter control plasmid pSico-SP146-Cre and CMVpromoter regulated negative control plasmid pSico were transfected293T andRAW264.7cell, regulation of Cre/LoxP system through the SP146promoter underthe circumstances, to observe the expression of green fluorescence identificationSP146promoter specificity and function of Cre recombinase.Results:1. After analysis the results through restriction Enzyme cutting and DNAsequencing, showed that four siRNA sequences were correctly inserted into plasmidpSicoR, and successfully constructed mTOR gene-specific siRNA lentiviral vectorpSM1, pSM2, pSM3and control siRNAexpression vector pSM4.2.293T cells packaged lentiviral expression vector pSM1/2/3/4, producelentiviral particles, detect the virus titer after concentrate the virus,the viral titer offour groups of recombinant lenti-pSM1/2/3/4were respectively6×106TU/ml,1×106TU/ml,4×106TU/ml,2.5×106TU/ml. Four kinds of recombinant lentiviralparticles infect RAW264.7cells, after48h, the group had observed lenti-pSM1/2/3/4bright green fluorescence, indicating a successful and effective recombinant lentivirusinfection RAW264.7cells.3. Real-time PCR and Western blot analysis of the results are consistent, bothconfirmed the mTOR mRNA and protein expression of lenti-pSM1group,lenti-pSM2group, lenti-pSM3group were inhibited. The inhibition rates of threeexperimental groups in mTOR mRNA expression levels were24.1%59.3%41.1%,protein expression levels with the control group (0.9567±0.021) and lenti-pSM4group (0.9200±0.030) of gray value were:0.9033±0.025,0.883±0.031,0.893±0.032,which lenti-pSM2group is the best group of the interference effect. 4. After analysis the results through restriction Enzyme cutting and DNAsequencing, showed that tthe sequence of Cre were correctly inserted into plasmidpBudCE4.1-SP146, and successfully constructed the recombinant plasmid pBudCE4.1-SP146-Cre.5. After analysis the results through colony PCR and DNA sequencing,confirmed that the sequence of macrophage specific promoter SP146and Cre genefragments were successfully inserted into the plasmid pSico, and constructedlentiviral vector pSico-SP146-Cre. The recombinant vector was transfected into293Tand RAW264.7cells,they was observed to have a strong green fluorescence in293Tcells, RAW264.7cells but almost no green fluorescence.Conclusion:1. Lentiviral expression vector pSM aimming at mTOR gene interferingsequence is successfully created.2. Completed lentivirus packaging and concentrated, successfully usingrecombinant lentiviral vector particles to produce virus, then the RAW264.7cellswere infected and screened mTOR shRNA2is the most effective interferencesequences.3. Lentiviral vector pSico-SP146-Cre of macrophage specific promoter SP146and recombinase Cre is successfully constructed.4. SP107has high specificity, which suggested the macrophage-specificexpression,it can be regulated Cre/LoxP system,and lay the foundation for thesubsequent experimental animal research level.