[Molecular cloning, protein expression and preparation of polyclonal antibody of ficolin-A].

AIM

To clone mouse ficolin-A cDNA, construct eukaryotic and prokaryotic expression vectors and prepare polyclonal antibody of ficolin-A.

METHODS

Full length of the ficolin-A cDNA fragment from the liver of newly born C57BL/6 mouse was amplified by RT-PCR by the use of GeneRacer kit. Then it was cloned into the eukaryotic vector pVAX-1 and prokaryotic vector pGEX-KG and expressed as a fusion protein in E.coli BL21 induced by IPTG. The expressed GST-ficolin-A fusion protein was purified via GST-Sepharose 4B Column and identified by SDS-PAGE and Western blot. The antibody against ficolin-A was prepared and its titer was identified.

RESULTS

The recombinant eukaryotic vector pVAX-1-ficolin-A and prokaryotic expression vector pGEX-KG-ficolin-A were successfully constructed. The recombinant GST-ficolin-A protein was overexpressed in E.coli. The relative molecular mass (M(r)) of the expressed product was identical with the predicted value. The antibody was prepared successfully.

CONCLUSION

We have successfully got the constructions of the recombinant eukaryotic vector pVAX-1-ficolin-A, prokaryotic expression vector pGEX-KG-ficolin-A and the purified recombinant ficolin-A protein in E.coli BL21. These will be helpful for the further investigation of the biological function of ficolin-A.