title: [Expression of EGF and SP domain proteins of MASP-2 and preparation of their polyclonal antibodies].
abstract: OBJECTIVE
  To clone and prokaryotically express the EGF and SP domain proteins of human mannan-binding lectin associated serine protease-2 (MASP-2),  and prepare their polyclonal antibodies and analyze their immunogenicity.
  
  
  METHODS
  The cDNA of human MASP-2 was transcripted reversely from total RNA extracted from human fetal liver tissue,  and then EGF and SP domain genes of MASP-2 were amplified from the cDNA by PCR. The EGF and SP fragment domain genes were cloned into pGEX-6P-2 expression vector and induced by IPTG to express GST fusion protein. The  BALB/c mice were immunized with the purified fusion proteins to generate polyclonal antibodies. The specificity of the polyclonal antibodies was analysed  with Western blotting,  and the titer was determined by indirect ELISA.
  
  
  RESULTS
  We successfully constructed the recombinant pGEX-6P-2-EGF and pGEX-6P-2-SP and induced the expressions of the GST-EGF and GST-SP fusion proteins. The specificity of the polyclonal antibodies was higher,  and had no cross reaction with other proteins. Indirect ELISA showed that the titer of anti-EGF antibody was more than 1:32 000 and anti-SP antibody was more than 1:40 000.
  
  
  CONCLUSIONS
  The polyclonal antibodies of the EGF and SP domain proteins we successfully obtained were of higher specificity and titer,  which  provides  the basis for further research on MASP-2.
authors.affiliation: Institute of Pathogen Biology and Immunology, Hebei North University, Zhangjiakou 075000, China. zhoufang5460@yahoo.com
authors.forename: Fang
authors.initials: F
authors.lastname: Zhou
authors.forename: Tian-jun
authors.initials: TJ
authors.lastname: Jia
authors.forename: Xiao-hui
authors.initials: XH
authors.lastname: Jia
authors.forename: Shu-yan
authors.initials: SY
authors.lastname: Liu
authors.forename: Xue-qing
authors.initials: XQ
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D006801:Humans; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000906:Antibodies; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases