Construction and identification of the eukaryotic expression vector carrying specific siRNA of LEDGF p52 gene
- VernacularTitle:LEDGFp52-siRNA真核表达载体的构建和鉴定
- Author:
Hai-Sheng, ZHAO
;
Yi, WANG
- Publication Type:Journal Article
- Keywords:
LEDGFp52;
RNA interference;
proliferation;
eukaryotic expression vector;
vector construction;
vector iden-tification
- From:
International Eye Science
2006;6(5):975-978
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To construct and identify LEDGFp52 eukaryotic expression vector for RNA interference.METHODS: Recombinants were designed and established by targeting gene LEDGFp52 and plasmid pGensil-1 based on LEDGFp52 cDNA sequences of Genomes. Two pairs of oligonucleotides were synthesized according to the Tuschl principle and inserted into plasmid pGenSil-I to generate siRNA eukaryotic expression vector. DH5α strains were transformed, plasmids were extracted, and recombinant vectors were identified by the restriction map and the sequence analysis. The cultured cells were transfected by the recombinant plasmid (pGensil-1-RNA. LEDGFp52-1). At 48 hours after transfection, the whole cell protein was extracted, and the protein level was detected using Western blotting with mouse anti-human LEDGFp52 monoclonal antibody.RESULTS: Recombinant plasmids completely concord with the designs by the restriction map and the sequence analysis,the proteinlevel of LEDGFp52 was down regulated at 48hours after transfecting pGensil-1- LEDGFp52-1 expression vector into HeLa cells, the recombinant eukaryotic expression vectors were successfully constructed.CONCLUSION: siRNA recombinant can be successfully constructed by RNAi technique to inhibit the expression of LEDGFp52.
