[Construction of "Pegfp-C1-pu6-dsrna" Recombinant for Human DNA Polymerase Beta RNA Interference].

OBJECTIVE:To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs).METHODS:According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing.RESULTS:The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment.CONCLUSION:The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.