title: [Prokaryotic expression   of  Nanog gene and preparation of anti-Nanog antibody].
abstract: OBJECTIVE
  To express Nanog fusion protein in Escherichia coli ( E.coli),  and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein.
  
  
  METHODS
  Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog. The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them. The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies. At last,  the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA,  Western blotting and immunocytochemical staining,  respectively.
  
  
  RESULTS
  The recombinant expression vector pET-32a-Nanog was successfully prepared,  transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli. After purification,  its purity was up to 97%. The titer of anti-Nanog antibodies was 1:32 000 in the immunized rabbit serum,  and exhibited a high specificity to Nanog protein.
  
  
  CONCLUSIONS
  The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.
authors: Department of Biology and Pharmacy Technology, Ningxia Collage of Vocation and Technology, Ningxia University, Yinchuan 750021, China. lijunamy@126.com
authors: Jun
authors: J
authors: Li
authors: Xiao-min
authors: XM
authors: Wang
authors: Zhong-ying
authors: ZY
authors: Dou
authors: Yong
authors: Y
authors: Li
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D000071317:Nanog Homeobox Protein; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000906:Antibodies; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D011993:Recombinant Fusion Proteins