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Construction of Packaging Plasmid pSANV2.0 of Adeno-associated Virus Vector(AAV)Carrying DREAM-targeting siRNA

CHEN Min1,XIANG Hong-bing2,TIAN Yu-ke1(1.Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;2.The Second Affiliated Hospital of Guangzhou Traditional Chinese Medicine University,Guangzhou 430070,China)  
[Purpose] To construct the packaging plasmid pSANV2.0-DREAMshRNA-EGFP of adeno-associated virus vector(AAV)carrying DREAM-targeting siRNA(small interfering RNA),which provide foundation for further study on the effect of DREAM gene for cancer pain treatment.[Methods ] We designed and synthesized two complementary oligonucleotide strands containing the small hairpin of DREAM,which was inserted into the EcoRⅠ and SalⅠ sites of pDC316-EGFP-U6 plasmid double digested by EcoRⅠ and SalⅠ.We got resultant plasmid pDC316-EGFP-DREAMshRNA-U6 and used it serving as PCR template to propagate DREAMshRNA-EGFP sgement,simutaneously EcoRⅠ and SalⅠ sites were introduced into PCR products.We ligated the PCR products and pSANV2.0 double digested by EcoRⅠ and SalⅠ to get resultant plasmid pSANV2.0-DREAMshRNA-EGFP.The liation product was transformed competence E.coli DH5.Positive clones were identified by PCR,enzyme digestion and sequencing.[Results] The result of PCR,restrictive enzyme digestion and gene sequencing confirmed that the inserted gene sequence and sites were correct and the plasmid pSANV2.0-DREAMshRNA-EGFP had been reconstructed successfully.[Conclusion] The packaging plasmid pSANV2.0-DREAMshRNA-EGFP of adeno-associated virus vector(AAV)carrying DREAM-targeting siRNA is successfully constructed.
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