[Preparation and identification of polyclonal antibodies against CUB1 and CUB2 functional domains from mannan-binding lectin-associated serine protease-2].
OBJECTIVE
To express CUB1 and CUB2 functional domain proteins of mannan-binding lectin-associated serine protease-2 (MASP-2) by prokaryotic expression system, and further prepare and identify the polyclonal antibodies against these two domains.


METHODS
The pGEX-6P-2 prokaryotic vector carrying the target gene CUB1 and CUB2 was used to prepare the recombinant protein GST-CUB1 and GST-CUB2. These two fusion proteins were purified with Glutathione SepharoseTM4B beads and then combined with Freund's adjuvant as antigens to immunize the BALB/c female mice aged 5 weeks for generating polyclonal antibodies. Subsequently, the specificity of the polyclonal antibodies was detected by Western blotting, and the titer was determined by indirect ELISA.


RESULTS
The fusion proteins GST-CUB1 and GST-CUB2 were successfully expressed and purified. Their polyclonal antibodies were gained from the BALB/c mice immunized with these two proteins. The polyclonal antibodies showed high specificity with no cross-reaction with other proteins. Indirect ELISA indicated that the titer of anti-CUB1 antibody was more than 1:32 000 and anti-CUB2 antibody was more than 1:16,000.


CONCLUSIONS
The polyclonal antibodies against GST-CUB1 and GST-CUB2 fusion proteins were obtained respectively with high titers and strong specificity.
Institute of Pathogen Biology and Immunology, School of Laboratory Medicine, Hebei North University, Zhangjiakou 075000, China.
Xiaodong
X
Han
Institute of Pathogen Biology and Immunology, School of Laboratory Medicine, Hebei North University, Zhangjiakou 075000, China.
Tianjun
T
Jia
Institute of Pathogen Biology and Immunology, School of Laboratory Medicine, Hebei North University, Zhangjiakou 075000, China.
Xiaohui
X
Jia
Institute of Pathogen Biology and Immunology, School of Laboratory Medicine, Hebei North University, Zhangjiakou 075000, China.
Ting
T
Li
Institute of Pathogen Biology and Immunology, School of Laboratory Medicine, Hebei North University, Zhangjiakou 075000, China.
Xia
X
Zhao
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D007114:Immunization; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000276:immunology*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000906:Antibodies; D011993:Recombinant Fusion Proteins; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases