Vegf Gene Transfection Experimental Study Of Bone And Soft Tissue Injury

Objective: To construct the vector pcDNA3.1 containing VEGF165 gene, detect its sequence and size, all of which lay a foundation for its application in bone defect and soft tissue injury.Methods: extract total RNA from the rabbit tissue by Trizol, design the primer according to the sequence of rabbit VEGF165 gene in Genebank. The sense primer: 5' GTG GAC ATC TTC CAG GAG TA 3' , the anti-sense primer: 5' CTT TGG TCT GCA TTC ACA 3' .the total size is 187bp. Prepare cDNA by inverse transcription and clone the gene by PCR. Combine the fragment to pMD18-T vector and transfer the product to competent cell, incubate the bacteria and plate them on LA plates. Pick up the colonies in LA medium and shake overnight in a 37℃ orbital shaker. Extract the vector by the kit, confirm the presence and orientation of the transgene by restriction analysis and detection sequence. Digest the pMD18-T vector and pcDNA3.1 vector in a 20-μl restriction by EcoR I and Hind III for 2 hours, gel purify the plasmid DNA and Combine the two plasmids with T4 DNA ligase in 10μl restriction, react overnight at 37℃. Retransfer the product to competent cell, incubate the bacteria and plate them on LA plates. Pick up the colonies in LA medium and shake overnight in a 37℃ orbital shaker. Extract the vector by the kit, confirm the presence and orientation of the transgene by restriction analysis and detection sequence.Results: The pcDNA3.1-VEGF165 vector was constructed successfully, the presence and orientation of the transgene was confirmed by detection sequence.Conclusion: The pcDNA3.1-VEGF165 vector is constructed successfully, which lays a foundation for the next experiments.