Construction Of Lentiviral Mediated RNA Interference Vector Of Suppressors Of Cytokine Signaling3Gene

Bronchial asthma is a chronic disease that occur at all ages and can seriously affect thequality of life，even threaten people’s health. Atopic dermatitis, allergic rhinitis, andbronchial asthma are allergic disorders characterized by a predominance of Th2polarisation,the synthesis of IgE, and mast cell and eosinophil-associated inflammation. The cytokineenvironment when the initial antigen stimulation determines the direction of helper T-celldifferentiation into Th1or Th2cells. The imbalance of Th1/Th2and Th2cell-based immuneresponses play an important role in the pathogenesis of allergic diseases. Suppressors ofcytokine signaling3(SOCS-3) is not only a feedback inhibitor of JAK/STAT pathway, butalso plays an important role in the regulation of cell differentiation and determine cell fate.SOCS-3highly expressed in Th2cells，it can be induced by many inflammatory oranti-inflammatory factor and affect Th2cells differentiation. Therefore, SOCS-3is apotential target for therapeutic strategies in allergic disorders.Objective: Construct mouse SOCS3RNA interference (RNAi) lentiviral vectors toprovide the experimental tools for the follow-up study.Methods: Filtered three corresponding target of mouse SOCS-3mRNA sequence byusing Whitehead Biomedical Research Center’s online RNAi design software.Complementary double-stranded oligonucleotide was designed to containing the desiredrestriction sites as well as sense and antisense target sequences, and then using BLAST forhomology search. This section was cloned into pLentilox3.7lentiviral vector plasmid thatdigested by restriction enzymes (HapI and XhoI), then we got reconstructive PLL3.7-SOCS3lentiviral vector plasmid. Reconstructive plasmids were transfected B16cell lines andobserved transfection in cells by fluorescence microscopy. After48hours cells wereharvested to extract total cellular RNA and detection SOCS-3expression by RT-PCR to filterout the best interference plasmid. The best plasmid and entiviral packaging plasmids werecotransfected into293T cells, collecting the supernatant of virus. Titer virus by serialdilution method.Result: We successful connected the target sequence with the vector, and confirmedvector was constructed successfully by double digestion with NotI and XbaI and sequencinganalysis. RT-PCR results showed that compared with control cells, three RNAi group wereinhibition SOCS3expression in different degrees. The strongest inhibitory effect were ingroup2. The titer of the lentiviral suspension were0.78×107/ml titered by serial dilution method.Conclusion: The reconstructive PLL3.7-SOCS3lentiviral vector can effectively inhibitthe expression of SOCS3in mouse. The SOCS3gene RNAi lentiviral vector was constructedsuccessfully.