Current HIV research · 2021
In vitro Anti-HIV-1 Activity of the Recombinant HIV-1 TAT Protein Along With Tenofovir Drug.
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Yadavar-Nikravesh Maryam-Sadat, Milani Alireza, Vahabpour Rouhollah, Khoobi Mehdi, Bakhshandeh Haleh, Bolhassani Azam
Аннотация
BACKGROUND: HIV-1 TAT protein is essential for the regulation of viral genome transcription. The first exon of TAT protein has a fundamental role in the stimulation of the extrinsic and intrinsic apoptosis pathways, but its anti-HIV activity is not clear yet.
METHODS: In the current study, we firstly cloned the first exon of the TAT coding sequence in the pET-24a expression vector and then protein expression was done in the Rosetta expression host. Next, the expressed TAT protein was purified by Ni-NTA column under native conditions. After that, the protein yield was determined by Bradford kit and NanoDrop spectrophotometry. Finally, the cytotoxicity effect and anti-Scr-HIV-1 activity of the recombinant TAT protein alone and along with Tenofovir drug were assessed by MTT and ELISA, respectively.
RESULTS: The recombinant TAT protein was successfully generated in E. coli, as confirmed by 13.5% SDS-PAGE and western blotting. The protein yield was ~150-200 μg/ml. In addition, the recombinant TAT protein at a certain dose with low toxicity could suppress Scr-HIV replication in the infected HeLa cells (~30%) that was comparable with a low toxic dose of Tenofovir drug (~40%). It was interesting that the recombinant TAT protein could enhance anti-HIV potency of Tenofovir drug up to 66%.
CONCLUSION: Generally, a combination of TAT protein and Tenofovir drug could significantly inhibit HIV-1 replication. It will be required to determine their mechanism of action in the next studies.
METHODS: In the current study, we firstly cloned the first exon of the TAT coding sequence in the pET-24a expression vector and then protein expression was done in the Rosetta expression host. Next, the expressed TAT protein was purified by Ni-NTA column under native conditions. After that, the protein yield was determined by Bradford kit and NanoDrop spectrophotometry. Finally, the cytotoxicity effect and anti-Scr-HIV-1 activity of the recombinant TAT protein alone and along with Tenofovir drug were assessed by MTT and ELISA, respectively.
RESULTS: The recombinant TAT protein was successfully generated in E. coli, as confirmed by 13.5% SDS-PAGE and western blotting. The protein yield was ~150-200 μg/ml. In addition, the recombinant TAT protein at a certain dose with low toxicity could suppress Scr-HIV replication in the infected HeLa cells (~30%) that was comparable with a low toxic dose of Tenofovir drug (~40%). It was interesting that the recombinant TAT protein could enhance anti-HIV potency of Tenofovir drug up to 66%.
CONCLUSION: Generally, a combination of TAT protein and Tenofovir drug could significantly inhibit HIV-1 replication. It will be required to determine their mechanism of action in the next studies.