Molecular Cloning And Expression Of MIF And Smad5 Gene From Hyriopsis Cumingii

In the study, MIF cDNA and Smad5 was cloned from from Hyriopsis cumingii, Rapid amplification of cDNA ends and nested PCR. Real-time quantitative PCR was used to analyze these genes expression characterization when injected Dextren, PGN, LPS, and Aeromonas hydrophila in tissues of hemocytes, hepatopancreas, musle, mantle, gills from H. cumingii.The full-length cDNA sequence of MIF was 723 bp, the 5’ untranslated region(UTR) and 3’ UTR were 69 bp and 309 bp, and contained a 345 bp open reading frame(ORF) coded for a 114-amina acids. The calculated molecular mass and isoelectric point(PI) of deduced protein were 13.83 KDa and 5.91, and the sequence of MIF has no signal peptide. The amino acid sequence had no Trp residues. The average hydrophobicity was 0.165 in structure. 4 Ser phosphorylation site were ser13 、 Ser59, Thr10, Thy37, respectively. The tertiary structure of MIF was composed of 3 structural domain, 6 alpha helix and 12 pieces of beta folding. Therefore, MIF is a trimer protein.The results of Real time quantitative PCR(qRT-PCR) analysis indicated that MIF gene was ubiquitously expressed in selected tissues, with the highest level in muscle, moderate level in gill, hepatopancreas, mantle, stomach, and the lowest level in hemocytes. MIF mRNA was significantly increased in hepatopancreas after the A. hydrophila and LPS challenged. It proved the MIF was important immune system in H. cumingii.In order to further inquire into the functions of MIF, PET-30 a prokaryotic expression system was constructed by double digestion and prokaryotic expression plasmid transformed into E.coli(DE3). The recombinant protein was successfully expressed after IPTG induction and was soluble existed in supernatant.The results confirmed that the recombinant protein was about 20 KD, which was in agreement with the predicted protein size.The complete cDNA sequence of Smad5 was 1627 bp, consisting of a 5’-terminal untranslated region(UTR) of 7 bp, a 3’-terminal UTR of 234 bp, containing an open reading frame(ORF) of 234 bp, encode 461 amino acids, containing signal signal peptide. The multiple sequence alignment analysis found that this gene has the two highly conserved Smad protein family activity sites(MH1 and MH2).