Title: Construction of a recombinant retroviral vector containing HIV-1 Tat gene and functional detection of the expressed Tat in target cells
Abstract: Objective To construct a recombinant retroviral vector containing HIV-1 Tat gene and evaluate the function of the expressed Tat in target cells. Methods HIV-1 Tat_ 101 gene was isolated from pEV plasmid by HindⅢ digestion and then cloned into expression plasmid LZRSpBMN-Z for constructing a recombinant retroviral expression plasmid named LZRS-Tat_ 101 . The construct of LZRS-Tat_ 101 was then transfected into packaging cell lines Phoenix (ФNX)using calcium phosphate which contained env and gal encoding for structural proteins while pol encoding for 3 enzymes (reverse transcriptase, protease and integrase) essential for retroviral integration and replication. The stable transfected cell lines was obtained by 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 above transiently and stably transfected ФNX respectively. Supernatants containing recombinant virus collected from transient and stable transfected cells were employed to infect 293 cells respectively and expressed Tat in 293 cells was tested by Western blot. Meantime, supernatants of infected 293 cells was added to HL3T1 cell (HeLa cell lines has contained a HIV-1-LTR/CAT reporter construct) to establish a co-culture system. After incubation for 72?h, protein was extracted from HL3T1 cells and used for CAT activity assay. Results ① After LZRS-Tat_ 101 was transfected into ФNX, the amount of expressed Tat in transient transfection cells was significantly more than that in stable transfection cells; ② Tat could be detected not only in 293 cells but also in the supernatants from 293 cells culture and the Tat in the supernatanst activated HIV-1 LTR promoter in HL3T1, resulting in the downstream high expression of CAT. Conclusion We conclude that construction of recombinant retrovirus LZRS-Tat_ 101 can express Tat protein in target cells and the expressed Tat is functionally active and shows a trans-activation activity.
Publication Year: 2003
Publication Date: 2003-01-01
Language: en
Type: article
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