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  • Title: [Molecular cloning, protein expression and preparation of polyclonal antibody of ficolin-A].
    Author: Zhou J, Sun P, Zhang XL.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2008 Sep; 24(9):881-3. PubMed ID: 18782522.
    Abstract:
    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.
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