Construction Of Recombinant Adeno-associated Virus Packaging System For Cytotoxic Gene-trichosanthin

Objective: Based on the regulatory function of mi RNA122 containing target gene,the negative regulation of endogenous mi RNA is applied to the negative regulation of transgenic expression,and the ectopic expression of transgenic fragments in the target gene-trichosanthin carrier is reduced and the packaging system of the cytotoxic gene r AAV vector is constructed.Methods:(1)Plasmid construction: first,the gene fragment was embedded in the plasmid driven by the chicken beta actin promoter and cytomegalovirus enhancer(CBAp)by molecular assembly technique,and the insertion site was located between the two terminal repeat sequences(ITR),and a series of target gene vectors were obtained for the preparation of r AAV.(2)Cell transfection: then the target gene was constructed into adeno-associated virus vector,and the constructed recombinant plasmid was introduced into the cell to detect the expression of the target protein.(3)double reporter gene detection: the expression level of the reporter gene was measured by Gaussia Luciferase and Firefly Luciferase,and a higher expression of mi R122 was established to enhance the ability to regulate the upstream gene of mir122 T.(4)packaging and purification of recombinant adeno-related virus: using q PCR,Western Blot and other techniques to compare a series of biological characteristics between the original cell line and the new cell line to detect the feasibility of packaging r AAV.Results: Endogenous and exogenous mi R-122 had tissue specificity and could regulate the expression of target gene.The HEK293-mir122-GLuc cell lines expressing mi R-122 and GLuc were isolated.The inhibitory effect of mir122 on the expression of target sequences was also suitable for HEK293-mir122-GLuc cell lines,and the growth rate and transfection efficiency were also applied.The HEK293-mir122-GLuc cells were superior to the original HEK293 cells,and the mir122 T sequence was inserted into the non-coding region of the cytotoxic gene expression frame to reduce the expression level of the toxic genes in the engineered cells.Finally,the virus packaging system was used to carry out the preparation and infection of the r AAV vector of the cytotoxic gene.Conclusion: The recombinant adeno-associated virus packaging system of cytotoxic gene trichosanthin was successfully constructed.