title: [The expression of humam sperm actin-like protein 7a and preparation of its polyclonal antibody].
abstract: OBJECTIVE
  To express and purify the recombinant human sperm actin-like protein 7a (ACTL7a) N terminal (1-70) in E.coli, and prepare the corresponding rabbit anti-ACTL7a polyclonal antibodies.
  
  
  METHODS
  We constructed the recombinant expression plasmid pGEX-6p-1-ACTL7a (1-70), transformed the plasmid into E.coli BL21 (DE3), and selected positive recombinant strain. The target protein was induced to express by IPTG and purified by nickel ions-chelating resin and glutathione sepharose 4B resin, and finally obtained by molecular sieve chromatography. The purified ACTL7a (1-70) protein were used to immunize New Zealand rabbits to prepare anti-ACTL7a polyclonal antibodies. The antibody titers were detected by ELISA, the antibody specificity by Western blotting, and the localization of ACTL7a in the spermatogenic cells of seminiferous tubule epithelium by immunofluorescence histochemistry.
  
  
  RESULTS
  The target protein ACTL7a (1-70) was successfully expressed by IPTG in E.coli, and the corresponding antiserum was obtained in the rabbits immunized with it. The titer of the antiserum reached 1:160 000 and it was proved with a good specificity.
  
  
  CONCLUSIONS
  ACTL7a was expressed in the constructed prokaryotic expression vector. After purification and immunization, high titer and specific anti-ACTL7a antibody was prepared in rabbits.
authors: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China. fjbnubio@126.com
authors: Jun
authors: J
authors: Fu
authors: Shiying
authors: S
authors: Miao
authors: Wei
authors: W
authors: Song
mesh_terms: D000199:Actins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification*; D000918:Antibody Specificity / Q000276:immunology; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D021381:Protein Transport; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D013094:Spermatozoa / Q000276:immunology / Q000378:metabolism*
chemical_list: C120133:ACTL7A protein, human; D000199:Actins; D000906:Antibodies; D011993:Recombinant Fusion Proteins