Construction and identification of recombinant adenovirus carrying human TIMP-1shRNA gene.
The aim of this study was to construct the recombinant adenovirus carrying human TIMP-1shRNA gene expression system for preliminary identification to lay the foundation for the further study of gene therapy. Using the Adeno-X system, the recombinant adenovirus plasmid pAdeno-X green fluorescent protein (GFP)-tissue inhibitor of metalloprotease (TIMP)-1 small hairpin (1shRNA) was constructed by including the target gene fragment of the TIMP-1shRNA shuttle plasmid pShuttle2-GFP-TIMP-1shRNA and the backbone plasmid pAdeno-X by homologous recombination in Escherichia coli. Recombinant plasmids were transfected into HEK293A cells to package the recombinant adenovirus rvAdeno-XGFP-TIMP-1shRNA. The recombinant adenovirus was identified by polymerase chain reaction, and the viral titer and infection efficiency were detected using GFP. Polymerase chain reaction and restriction endonuclease digestion demonstrated that rvAdeno-XGFP-TIMP-1shRNA had been successfully constructed, which has a strong ability to infect the kidney. The TIMP-1shRNA adenovirus expression vector was successfully constructed using homologous recombination methods.
The First Hospital of Dalian Medical University, Division of Nephrology, Dalian, China.
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Sun
The First Hospital of Dalian Medical University, Division of Nephrology, Dalian, China huaxiecn@126.com.
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Xie
The First Hospital of Dalian Medical University, Division of Nephrology, Dalian, China.
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Lin
The First Hospital of Dalian Medical University, Division of Nephrology, Dalian, China.
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Feng
The First Hospital of Dalian Medical University, Division of Nephrology, Dalian, China.
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Liu
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D048430:Cell Shape; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000378:metabolism*; D051381:Rats; D011995:Recombination, Genetic; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics*; D014162:Transfection
D034741:RNA, Small Interfering; C560822:TIMP1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins