ISSN: 2423-6241
Winter
Vol. 2 No. 1 (2016)
Cloning, expression and library construction for HIV-1 Tat Protein
Archives of Medical Laboratory Sciences,
Vol. 2 No. 1 (2016),
https://doi.org/10.22037/amls.v2i1.13688
Abstract
Background: Designing novel therapeutic agents has been a critical challenge for HIV disease.
Materials and Methods: In current study a DNA sequence which was encoded the Tat protein was synthesized and inserted in pET 28 vector. Vector was cloned in BL21-DE3 E. coli and cultured in TB media. After protein expression, recombinant Tat protein was purified by NTA affinity chromatography. The Tat purified protein efficiency and confirmed by SDS-PAGE and Western blot, respectively. We were immunized the camel against HIV-1 Tat recombinant protein to made a camelid antibody library. Total RNA was extracted from camel lymphocytes and VHH fragments synthesized and amplified using RT-PCR and Nested- PCR methods by special primers.
Results: The 350- 450 bp VHH gene fragment was produced by RT-PCR and Nested- PCR and extracted from agarose gel 1%. Then gel extraction was performed and pure fragments were inserted in HEN-4 vector by T4 DNA ligase.
Conclusion: The library can be applied for biopanning and isolation of nanobody against HIV-1 Tat Protein. Nanobody small size may be a useful drug for treatment of HIV disease because give them the potency of the recognizing the cryptic epitopes of tat and neutralized the virus.
- HIV-1
- Tat Protein
- VHH
- Nanobody
How to Cite
References
Debaisieux S, Rayne F, Yezid H, Beaumelle B. The Ins and Outs of HIV‐1 Tat. Traffic. 2012;13(3):355-63.
Gharu L, Marcello A. Overcoming the Transcriptional Block: The HIV-1 Tat Auxiliary Protein. 2015.
Karn J, Stoltzfus CM. Transcriptional and posttranscriptional regulation of HIV-1 gene expression. Cold Spring Harborperspectives in medicine. 2012;2(2):a006916.
McCauley MJ, Rouzina I, Hoadley K, Gorelick RJ, Musier-Forsyth K, Williams MC. Specific Binding of the Nucleocapsid Protein Transforms the Folding Landscape of the HIV-1 TAR RNA Hairpin. Biophysical Journal. 2015;108(2):398a.
Lu J, Nguyen L, Zhao L, Xia T, Qi X. A Cyclic Mimic of HIV Tat Differentiates Similar TAR RNAs on the Basis of Distinct Dynamic Behaviors. Biochemistry. 2015;54(23):3687-93.
Dhamija N, Choudhary D, Ladha JS, Pillai B, Mitra D. Tat predominantly associates with host promoter elements in HIV‐1‐infected T‐cells–regulatory basis of transcriptional repression of c‐Rel. FEBS journal. 2015;282(3):595-610.
Paz S, Lu ML, Takata H, Trautmann L, Caputi M. SRSF1 RNA Recognition Motifs Are Strong Inhibitors of HIV-1 Replication. Journal of virology. 2015;89(12):6275-86.
Mbonye U, Karn J. Transcriptional control of HIV latency: cellular signaling pathways, epigenetics, happenstance and the hope for a cure. Virology. 2014;454:328-39.
Colin L, Verdin E, Van Lint C. HIV-1 chromatin, transcription, and the regulatory protein Tat. Human Retroviruses: Methods and Protocols. 2014:85-101.
Buonaguro L, Buonaguro F, Giraldo G, Ensoli B. The human immunodeficiency virus type 1 Tat protein transactivates tumor necrosis factor beta gene expression through a TAR-like structure. Journal of virology. 1994;68(4):2677-82.
Planès R, Haij NB, Leghmari K, Serrero M, BenMohamed L, Bahraoui E. HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes. Journal of virology. 2016;90(13):5886-98.
Shearer GM, Clerici M. Early T-helper cell defects in HIV infection. Aids. 1991;5(3):245-54.
Cullen BR. HIV-1 auxiliary proteins: making connections in a dying cell. Cell. 1998;93(5):685-92.
Arter J, Wegner M. Transcription factors Sox10 and Sox2 functionally interact with positive transcription elongation factor b in Schwann cells. Journal of neurochemistry. 2015;132(4):384-93.
Howcroft TK, Strebel K, Martin MA, Singer DS. Repression of MHC class I gene promoter activity by two-exon Tat of HIV. Science. 1993;260(5112):1320-2.
Chiozzini C, Toschi E. HIV-1 Tat and Immune Dysregulation in AIDS Pathogenesis: A Therapeutic Target. Current drug targets. 2016;17(1):33-45.
Poggi A, Zocchi MR. HIV-1 Tat triggers TGF-β production and NK cell apoptosis that is prevented by pertussis toxin B. Journal of Immunology Research. 2006;13(2-4):369-72.
Bellino S, Tripiciano A, Picconi O, Francavilla V, Longo O, Sgadari C, et al. The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study. Retrovirology. 2014;11(1):1.
Paris JJ, Singh HD, Ganno ML, Jackson P, McLaughlin JP. Anxiety-like behavior of mice produced by conditional central expression of the HIV-1 regulatory protein, Tat. Psychopharmacology. 2014;231(11):2349-60.
Cook JA, August A, Henderson AJ. Recruitment of phosphatidylinositol 3-kinase to CD28 inhibits HIV transcription by a Tat-dependent mechanism. The Journal of Immunology. 2002;169(1):254-60.
Bedoya LM, Beltrán M, Sancho R, Olmedo DA, Sánchez-Palomino S, del Olmo E, et al. 4-Phenylcoumarins as HIV transcription inhibitors. Bioorganic & medicinal chemistry letters. 2005;15(20):4447-50.
Alirezapour B, Rajabibazl M, Rasaee MJ, Omidfar K. Production and characterization of recombinant scFv against digoxin by phage display technology. Monoclonal antibodies in immunodiagnosis and immunotherapy. 2013;32(3):172-9.
Mediouni S, Watkins JD, Pierres M, Bole A, Loret EP, Baillat G. A monoclonal antibody directed against a conformational epitope of the HIV-1 trans-activator (Tat) protein neutralizes cross-clade. Journal of Biological Chemistry. 2012;287(15):11942-50.
Rissiek B, Koch-Nolte F, Magnus T. Nanobodies as modulators of inflammation: potential applications for acute brain injury. Frontiers in cellular neuroscience. 2014;8:344.
McCoy LE, Groppelli E, Blanchetot C, de Haard H, Verrips T, Rutten L, et al. Neutralisation of HIV-1 cell-cell spread by human and llama antibodies. Retrovirology. 2014;11(1):1.
Forsman A, Beirnaert E, Aasa-Chapman MM, Hoorelbeke B, Hijazi K, Koh W, et al. Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. Journal of virology. 2008;82(24):12069-81.
Forsman AMM. Characterisation of llama antibody fragments able to act as HIV-1 entry inhibitors: UCL (University College London); 2009.
Vercruysse T, Pardon E, Vanstreels E, Steyaert J, Daelemans D. An intrabody based on a llama single-domain antibody targeting the N-terminal α-helical multimerization domain of HIV-1 Rev prevents viral production. Journal of Biological Chemistry. 2010;285(28):21768-80.
- Abstract Viewed: 536 times
- PDF Downloaded: 441 times
