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Prokaryotic Expression and Activity Detection of Ad5/Ad35 Fiber Gene |
ZHANG Wen-feng1,2, ZHANG Qiong-yu3, BO Hua-ben1,2, SHAO Hong-wei1,2, LI Xiao-cheng1,2, WANG Teng1,2, HUANG Shu-lin1,2 |
1. School of Life Science & Biopharmacology, Guang Dong Pharmaceutical University, Guanzhou 510006, China; 2. Guang Dong Provincial Key Laboratory of Biotechnology Candidate Drug Research, Guangzhou 510006, China; 3. Department of Basic Medical Science, Yongzhou Vocational Technical College, Yongzhou 425100, China |
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Abstract Aim: To construct the prokaryotic expression vector for Ad5 fiber and Ad35 fiber genes, purify proteins and study the activity. Methods: Recombinant plasmid pET28a-Ad5 fiber and pET28a-Ad35 fiber were transformed into E.coli BL21(DE3) and the proteins expression were induced with IPTG. The expressed proteins Ad5 fiber and Ad35 fiber were purified by Ni2+ affinity chromatography and identified by SDS-PAGE. The bioactivity of proteins Ad5 fiber and Ad35 fiber were certificated by competitive binding method. Results: The proteins Ad5 fiber and Ad35 fiber can be detected in the supernatant. The proteins Ad5 fiber specifically inhibited the infectivity of the Ad5 virus in a dose-dependent manner, while had no effect on the Ad5F35-mediated gene transfer. The proteins Ad35 fiber blocked the Ad5F35-mediated gene transfer in a dose-dependent manner and had no effect on the infectivity of the Ad5 virus in competition experiments. Conclusion: Recombinant vectors Ad5 fiber and Ad35 fiber were constructed successfully. The proteins Ad5 fiber and Ad35 fiber could specifically bind with different receptors. The study will lay a foundation for the investigation on the mechanism of Ad5F35 infection.
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Received: 06 September 2013
Published: 25 December 2013
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[1] Clay T M, Custer M C, Sachs J, et al. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J Immunol, 1999, 163: 507-513. [2] Morgan R A, Dudley M E, Wunderlich J R, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science, 2006, 314: 126-129. [3] Johnson L A, Morgan R A, Dudley M E, et al. Gene therapy with human and mouse T cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood, 2009, 114: 535-546. [4] Uckert W, Becker C, Gladow M, et al. Efficient gene transfer into primary human CD8+ T lymphocytes by MuLV-10A1 retrovirus pseudotype. Hum Gene Ther, 2000, 11: 1005-1014. [5] Uckert W, Schumacher T N. TCR transgenes and transgene cassettes for TCR gene therapy: status in 2008. Cancer Immunol Immunother, 2009, 58: 809-822. [6] Nicklin S A, Wu E, Nemerow G R, et al. The influence of adenovirus fiber structure and function on vector development for gene therapy. Mol Ther, 2005, 12: 384-393. [7] Tomko R P, Xu R, Philipson L. HCAR and MCAR: the human and mouse cellular receptors for subgrou PC adenoviruses and grou PB coxsackieviruses. Proc Natl Acad Sci USA, 1997, 94: 3352-3356. [8] Gaggar A, Shayakhmetov D M, Lieber A. CD46 is a cellular receptor for grou PB adenoviruses. Nat Med, 2003, 9:1408-1412. [9] Schroers R, Hildebrandt Y, Hasenkamp J, et al. Gene transfer into human T lymphocytes and natural killer cells by Ad5/F35 chimeric adenoviral vectors. Ex PHematol, 2004, 32: 536-546. [10] Shayakhmetov D M, Papayannopoulou T, Stamatoyannopoulos G, et al. Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector. J Virol, 2000, 74: 2567-2583. [11] Knan-Shanzer S, Marloes J M, Watering V D, et al. Endowing human adenovirus serotype 5 vectors with fiber domains of species B greatly enhances gene transfer into human mesenchymal stem cells. Stem Cells, 2005, 23:1598-1607. |
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