|
|
Preparation and Evaluation of Polyclonal Antibodies of Apoptin |
WANG Chun-hui1, WANG Jian-song1, WANG Wen-ju2, ZHAN Hui1, LI Hong-jun2, YAN Ru-ping1, XU Hong-yi1 |
1. Department of Urology, The Second Affiliated Hospital of Kunming Medical University, Kunming 650101, China;
2. Institute of Medical Biology, Chinese Academy of Medical Science ﹠ Peking Union Medical School, Kunming 650118, China |
|
|
Abstract Apoptin is coded by the VP3 gene from chicken anemia virus (CAV), and the protein can induce apoptosis of a large variety of tumor cells. The preparation of polyclonal antibodies specific to VP3 protein was explored. The VP3 gene was obtained from the eukaryotic expression vector pcDNA3.0-VP3. And then, VP3 gene was cloned into the prokaryotic expression vector pET8a. The prokaryotic expression vector pET8a-VP3 was employed to express VP3 protein in E.coli BL21 which was induced by IPTG, and the protein was extracted from the cell and purified. The purified VP3 protein was applied to immunize the New Zealand rabbits combined with the Freunds complete adjuvant or incomplete adjuvant by multi-points subcutaneous injection. ELISA test was performed to measure the titer of the antibody after immunization. Two days after the measurement the whole blood was harvested by cardiac puncture and the serum was separated from the whole blood. The immunglobin IgG was purified by Protein A method from the antiserum. The final purified antibody titers were up to 1 ∶ 243 000. The immunological evaluation of the polyclonal antibody specific binding was assayed by using recombinant adeno-associated virus rAAV-VP3 infected different cell lines. First, it was used to detect the VP3 gene expression by immunofluorescence in human bladder cancer cells T24 and EJ, also in Vero cells. Apoptin was observed in T24, EJ cells, mainly located in the nucleus, whereas in Vero cells were localized in the cytoplasm. Secondly, the specific binding of the purified antibodies to VP3 protein in different human bladder cancer cells were detected by Western blotting. The results have shown that the polyclonal antibody have displayed good effectiveness and binding specificity to apoptin, which will provide a foundation for further clarify the molecular mechanisms of anti-tumor effect and biological characteristics of apoptin.
|
Received: 14 March 2011
Published: 25 July 2011
|
|
Fund: Supported by the National Natural Science Foundation of China (50774102) and Major State Basic Research Development Program of China (2010CB630900) |
|
|
[1] Noteborn M H. Apoptin acts as a tumor-specific killer: potentials for an anti-tumor therapy. Cell Mol Biol (Noisy-le-grand), 2005, 51(1):49-60.
[2] Backendorf C, Visser A E, de Boer A G, et al. Apoptin: therapeutic potential of an early sensor of carcinogenic transformation. Annu Rev Pharmacol Toxicol, 2008, 48(2): 143-169.
[3] Rohn J L, Noteborn M H. The viral death effector Apoptin reveals tumor-specific processes. Apoptosis, 2004, 9(3): 315-322.
[4] 王文举, 孙茂盛, 严敏, 等. Neurturin嵌合神经营养因子重组腺病毒的构建及体外活性测定. 中国生物工程杂志, 2010, 30(9): 7-12. Wang W J, Sun M S, Yan M, et al. China Biotechnology, 2010, 30(9): 7-12.
[5] Schoop R A, Kooistra K, Baatenburg De Jong R J, et al. Bcl-xL inhibits p53-but not apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line. Int J Cancer, 2004, 109(1): 38-42.
[6] Noteborn M H. Chicken anemia virus induced apoptosis: underlying molecular mechanisms. Vet Microbiol, 2004, 98(2):89-94.
[7] Danen-Van Oorschot A A, Zhang Y H, Leliveld S R, et al. Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis. J Biol Chem, 2003, 278(30): 27729-27736.
[8] Rohn J L, Zhang Y H, Aalbers R I, et al. A tumor-specific kinase activity regulates the viral death protein Apoptin. J Biol Chem, 2002, 277(52): 50820-50827.
[9] Leliveld S R, Dame R T, Mommaas M A, et al. Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA. Nucleic Acid Res, 2003, 31(16): 4805-4813.
[10] Leliveld S R, Dame R T, Rohn J L, et al. Apoptins functional N-and C-termini independently bind DNA. FEBS Lett, 2004, 557(1-3): 155-158.
[11] Liu X, Zeidan Y H, Elojeimy S, et al. Involvement of sphingolipids in apoptin-induced cell killing. Mol Ther, 2006, 14(5):627-636.
[12] Wadia J S, Wagner M V, Ezhevsky S A, et al. Apoptin/VP3 contains a concentration-dependent nuclear localization signal(NLS), not a tumorigenic selective NLS. J Virol, 2004, 78(11): 6077-6078.
[13] 王春晖, 王剑松, 詹辉, 等. 改良型TAT-凋亡素体外抗人膀胱肿瘤活性的研究. 中国肿瘤临床, 2011, 38(5): 246-249. Wang C H, Wang J S, Zhan H, et al. Chinese Journal of Clinical Oncology, 2011, 38(5): 246-249.
[14] Heilman D W, Teodoro J G, Green M R. Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies. J Virol, 2006, 80(15): 7535-7545.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|