[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.

CONCLUSION

The polyclonal antibodies against GST-CUB1 and GST-CUB2 fusion proteins were obtained respectively with high titers and strong specificity.