High-level Expression, Polyclonal Antibody Preparation Of Mdfic Protein And Its Tissue Distribution Analysis

Mdfic(MyoD family inhibitor domain containing) gene, was a newly discovered gene in recent years. It encode a protein containing the I-mfa domain with unknown function. Mdfic was shown to interact with Rhox5 in our previous study. It suggested that Mdfic might be involved in the transcription that was regulated by Rhox5. Thus, in turn, participateds in the embryonic development process and the differentiation regulation together with Rhox5 protein. In order to further investigate the function of the Mdfic, the Mdfic recombinant protein was expressed and purified using the prokaryotic expression system. Polyclonal anti-Mdfic antiserum was subsequently generated for future studies.Object: Overexpression of GST-Mdfic protein in E. coli; getting specific polyclonal antiserum of Mdfic protein; characterize the subcellular distribution of Mdfic; quantitate the Mdfic expression leveling different mouse tissue.Method: Sequence analysis showed that Mdfic, a mouse orthologue of HIC(Human I-mfa domain containing protein), belonged to I-mfa superfamily. The Mdfic ORF was cloned into E. coli expression vector pEGX-4T-3 and the GST fusion protein was expressed in BL21. GST-Mdfic fusion protein was purified by affinity chromatography and was then used to immunize male New Zealand white rabbit by intradermal injection. The titer and the specificity of the rabbit antiserum were shown by ELISA and Western blotting, respectively. The antiserum was then used in localizing Mdfic by immunofluorescence. The expressions of Mdfic in different mouse tissues were studied by real-time PCR.Result: Sequence analyze of Mdfic indicated that the Mdfic gene was mouse gene orthologue of HIC (Human I-mfa domain containing protein), belonging to the I-mfa domain family. The pGEX-4T-3-Mdfic E. coli expression plasmid was constructed and expressed successfully; the GST-Mdfic was purified with affinity chromatography and the purified product was ultrafiltered to desalt. The polyclonal antiserum was collected from the immunized rabbit. Its titer was 1:640, 000 and could specifically recognize endogenous and exogenous Mdfic protein, according to the result of ELISA and Western blotting, respectively. The indirect immunofluorescence using this antiserum indicated that Mdfic was distributed in both cytoplasm and nuclear. Real Time PCR demonstrated Mdfic was highly expressed in the livers, spleen, kidney and testicle in adult mice.Conclusion: Mdfic specific polyclonal antiserum was collected and used successfully. In localizing subceUular Mdfic by immunofluorescence; different levels of expression of Mdfic were observed in different mouse tissues.