Construction of Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells

摘要

Objective: To construct recombinant retroviral vector containing HIV-1 Tat gene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) gene was recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z to construct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method of calcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell lines Phoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene coding for 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integration and replication . The stable transfected cell lines was obtained using puromycin to screen for more than 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expression level of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. The supematants containing recombinant virus collected from transient and stable transfected cells were employed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Western blot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which were Hela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system. After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activity assay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat in transient transfection cells was significantly higher than that in stable transfection cells; Tat could be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tat in the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CAT located at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS- Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally active and can really exhibit the ability to activate transcription.