Abstract:

The specific amino acid sequence(104123) in βdomain of pVHL is sufficient to inhibit the growth and invasion of renal cell carcinoma（RCC）. To express this peptide for a gene therapy of RCC by using minigene, we constructed and identified the recombinant plasmid expression box, including NT4 signal peptide, tag and cell penetrating peptides. MethodsBy means of asymmetrical primer/template, fragments encoding βdomain of pVHL(the VHL gene product) and TAT6×His were gained, which included Nae I/Eco72I and Eco72I/BamH I restriction enzyme sites. Then the fragments were cloned into vector pGEMT easy. The positive clone was identified by restriction enzymes, and then the cloned amplified fragments were sequenced by a dideoxymediated chaintermination method. The cloned TAT6×HisVHLβ cDNA was compared with the GeneBank sequence by DNASIS. After being digested by Nae I and BamH I, the TAT6×HisVHLβ cDNA fragment was cloned into recombinant vector pBV220NT4, which was also digested by Nae I and BamH I. The constructed recombinant plasmid pBV220NT4TAT6×HisVHLβ was obtained. ResultsThe sequences of TAT6×His and VHLβ cDNA artificially synthesized and analyzed by digestion were consistent with the published results. After analyzing ORF of the cloned TAT6×HisVHLβ cDNA by DNASIS, we found that amino acids encoded by the cloned TAT6×HisVHLβ cDNA were also identical to the published results. ConclusionThe recombinant plasmid expressing TAT cell penetrating peptides and βdomain of pVHL was constructed. Then prokaryotic expressive vector pBV220NT4TAT6×HisVHLβ was constructed by a routine molecular biological method. These results lay a foundation for further research of fusion gene to treat RCC.

Key words:  VHLβdomain RCC supressor peptide； Fusion peptide； Minigene； Kidney neoplasms