关键词: RNA干扰, 核小体结合蛋白, 慢病毒

Abstract: Objective: To construct and identify a lentiviral vector harboring RNAi sequence targeting NSBP1 gene. Methods: Three siRNA targeting the NSBP1 mRNA were designed, and the pGCSIL-GFP-NSBP1 lentivirus vectors were constructed, confirmed by DNA sequencing. A total of 293T cells were co-transfected with pGCSIL-GFP-NSBP1，pHelper1.0 and pHelper2.0 for the virus stocks produced, the titer of the virus was tested. After lentivirus were transfected into DU145 cells, Western-blot and MTT methods were used to determine the expression and biological activity of NSBP1 gene, the cells were transplanted into nude mice, then inhibitive effect was observed. Results: PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting the human NSBP1 gene was successfully inserted into the lentiviral vector. The titer of the recombinant lentiviral vector as 2×108TU/ml. NSBP1 protein expression level in transfected cells was significantly decreased and growth rate of cells transfected with lentivirus was decreased by MTT assay, the downregulation of NSBP1 reduced growth rate of transplantated tumor, whereas tumorgenicity was not influenced. Conclusion: The construction of the lentiviral vector of NSBP1 has been successfully prepared and NSBP1 plays an important regulatory role in androgen-independent prostate cancer cell proliferation.

Key words: RNA interference, nucleosomal binding protein, lentivirus, westernblot, MTT