Abstract The transcription factor Nanog plays a core role to maintain the self-renewal and pluripotency of stem cells. In order to study the regulatory function of pig (Sus scrofa) Nanog in pluripotent networks, the recombinant Nanog protein was used to generate the mouse anti-Nanog polyclonal antibody. Firstly, the CDS of Nanog cloned from porcine kidney was connected into pET32a vector to construct the recombinant expression vector pET32a-Nanog. Next, the pET32a-Nanog was transformed into Escherichia coli Rosseta (DE3) and Nanog expression was induced by isopropyl-β-d-thiogalactoside (IPTG). After optimizing the induction time, temperature and IPTG concentrations, the recombinant Nanog protein (57 kD) was obtained. The Nanog protein was purified and then used as the antigen to immunize C57BL/6 mice (Mus musculus). After 6 weeks, the positive antiserum was collected, and the antibody titer and specificity were detected by Western blot and immunofluorescence assay. The results showed that the recombinant Nanog could be highly expressed under the induction conditions of 28 ℃, 8 mmol/L IPTG and 2.5 h; the generated polyclonal anti-Nanog antibody could react specifically with Nanog generated from porcine induced pluripotent stem cells, suggesting that the polyclonal antibody can provide a powerful tool for porcine pluripotent stem cell research.
YU Zhuan-Zhen,LIU Xiao-Peng,CUI Yi, et al. High Level Expression, Preparation and Application of Anti-Nanog Polyclonal Antibody of Porcine (Sus scrofa) Nanog Protein [J]. , 2016, 24(1): 35-43.
[1]A., Schreier, L. L., Talbot, N. C..Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast during in vitro culture[J].Molecular Reproduction and Development, 2008, 75(3):450-463 [2]Booth, H A. F., Holland, P. W..Eleven daughters of NANOG[J].Genomics, 2004, 84(2):229-238 [3]Chambers, I, Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., Smith, A..Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells[J].Cell, 2003, 113(5):643-655 [4]Chang, D F., Tsai, S. C., Wang, X. C., Xia, P., Senadheera, D., Lutzko, C.Molecular characterization of the human NANOG protein[J].Stem Cells, 2009, 27(4):812-821 [5]Clark, E D. B..Protein refolding for industrial processes[J].Current opinion in biotechnology, 2001, 12(2):202-207 [6]Darr, H , Mayshar, Y., Benvenisty, N. .Overexpression of NANOG in human ES cells enables feeder-free growth while inducing primitive ectoderm features. [J].Development, 2006, 133(6):1193-1201 [7]Esteban, M, Xu, J., Yang, J., Peng, M., Qin, D., Li, W., Jiang, Z., Chen, J., Deng, K., Zhong, M.Generation of induced pluripotent stem cell lines from Tibetan miniature pig [J].Journal of Biological Chemistry, 2009, 284(26):17634-17640 [8]Evans, M J., Kaufman, M. H..Establishment in culture of pluripotential cells from mouse embryos[J].nature, 1981, 292(5819):154-156 [9]Ezashi, T, Telugu, B., Alexenko, A., Sachdev, S., Sinha, S., Roberts, R.Derivation of induced pluripotent stem cells from pig somatic cells[J].Proceedings of the National Academy of Sciences, 2009, 106(27):10993-10998 [10]Guo, G, Yang, J., Nichols, J., Hall, J. S., Eyres, I., Mansfield, W., Smith, A.Klf4 reverts developmentally programmed restriction of ground state pluripotency[J].Development, 2009, 136(7):1063-1069 [11]Hall, V.Porcine embryonic stem cells: a possible source for cell replacement therapy[J].Stem cell reviews, 2008, 4(4):275-282 [12]Hart, A H., Hartley, L., Ibrahim, M., Robb, L. .Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human[J].Developmental dynamics, 2004, 230(1):187-198 [13]Hyslop, L, Stojkovic, M., Armstrong, L., Walter, T., Stojkovic, P., Przyborski, S., Herbert, M., Murdoch, A., Strachan, T., Lako, M..Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages[J].Stem Cells, 2005, 23(8):1035-1043 [14]Kumar, B, Jin, H., Kim, J., Ock, S., Hong, Y., Balasubramanian, S., Choe, S., Rho, G. .Differential gene expression patterns in porcine nuclear transfer embryos reconstructed with fetal fibroblasts and mesenchymal stem cells[J].Developmental dynamics, 2007, 236(1):435-446 [15]Li, J, Pu, M., Hirasawa, R., Li, B., Huang, Y., Zeng, R., Jing, N., Chen, T., Li, E., Sasaki, H. .Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog[J].Molecular and cellular biology, 27(24):: 8748–8759., 2007, 27(24):8748-8759 [16]Li, Z-Y., Liu, C.-P., Zhu, L.-Q., Jing, G.-Z., Zhou, J.-M. .The chaperone activity of trigger factor is distinct from its isomerase activity during co-expression with adenylate kinase in Escherichia coli[J].FEBS Lett, 2001, 506(2):108-112 [17]Mitsui, K, Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M., Yamanaka, S..The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J].Cell, 2003, 113(5):631-642 [18]Oh, J-H., Do, H.-J., Yang, H.-M., Moon, S.-Y., Cha, K.-Y., Chung, H.-M., Kim, J.-H..Identification of a putative transactivation domain in human Nanog[J].Exp Mol Med, 37(3):250-254., 2005, 37(3):250-254 [19]Pan, G, Pei, D..The stem cell pluripotency factor NANOG activates transcription with two unusually potent subdomains at its C terminus[J].Journal of Biological Chemistry, 2005, 280(2):1401-1407 [20]Silva, J, Nichols, J., Theunissen, T., Guo, G., van, O. A., Barrandon, O., Wray, J., Yamanaka, S., Chambers, I., Smith, A..Nanog is the gateway to the pluripotent ground state[J].Cell, 2009, 138(4):722-737 [21]Theunissen, TW., Oosten, A. L. v., Castelo-Branco, G., Hall, J., Smith, A., Silva, J. C. R. .Nanog Overcomes Reprogramming Barriers and Induces Pluripotency in Minimal Conditions[J].Current Biology, 2011, 21(1):65-71 [22]West, FD., Terlouw, S. L., Kwon, D. J., Mumaw, J. L., Dhara, S. K., Hasneen, K., Dobrinsky, J. R., Stice, S. L. .Porcine induced pluripotent stem cells produce chimeric offspring[J].Stem cells and development, 2010, 19(8):1211-1220 [23]Wu, Z, Chen, J., Ren, J., Bao, L., Liao, J., Cui, C., Rao, L., Li, H., Gu, Y., Dai, H. .Generation of pig-induced pluripotent stem cells with a drug-in-ducible system[J].Chinese Journal of Transplanation(Electronic Edition), 2009, 1(1):46-54 [24]Yang, F, Zhang, J., Liu, Y., Cheng, D., Wang, H..Structure and functional evaluation of porcine NANOG that is a single-exon gene and has two pseudogenes[J].The international journal of biochemistry & cell biology, 2014, 5(9):142-152 [25]Zhu, J, King, T., Dobrinsky, J., Harkness, L., Ferrier, T., Bosma, W., Schreier, L., Guthrie, H., DeSousa, P., Wilmut, I. .In vitro and in vivo developmental competence of ovulated and in vitro matured porcine oocytes activated by electrical activation[J].Cloning Stem Cells, 2003, 5(4):355-365
