Melecular Cloning,expression And Construction Of Wound Repair Model Of Matrix Metalloproteinase Gene From Hyriopsis Cumingii

The freshwater mussel,which is one of great economical importance “pearl bivalves” in the aquaculture industry of China,has been suffering serious problems due to the outbreak of diseases,increased the rate of spitting nuclear and even caused the death of pearl bivalves.Thus,understanding the mechanism of molecular immunity and wound repair have the important significance of theory and practice.In this study,Matrix metalloproteinases-1(MMP-1)and Matrix-metalloproteinases-19(MMP-19)cDNA were cloned from H.cumingii using rapid amplification of cDNA ends.The expression of MMP1,19 mRNA was measured by real-time fluorescence quantitative PCR in five organizations of a health mussel such as hemocytes,hepatopancreas,muscle,mantle and gills.In addition,both genes were detected in blood and hepatopancreas after Aeromonas hydrophila and Peptidoglycan stimulation.The model of nucleus inserting operation trauma was established,and injury repairing was assessed by the expression levels in different time after trauma.Two genes were prokaryotic expressed by prokaryotic expression technique.The inhibitory activity of recombinant protein against metalloproteinases was determined.The MMP1 cDNA was 1822 bp in length and contained a 5’ untranslated region(UTR)of 31 bp,a 3’ UTR of 258 bp and 1533 bp open reading frame(ORF)encoding a total of 510 amino acid residues.Analysis of SignalP 4.0 by Expasy online site found the molecular weight of the predicted MMP1 was 58.28 kDa,with the calculated PI being 9.27,SignalP program analysis showed that it had signal peptide.The length cDNA sequence of the cloned MMP19 was 2130 bp and contained a 1569 bp open reading frame(ORF)encoding 522 amino acid residues and had 29 bp of 5 ’ untranslated region(UTR)and a 3’ UTR of 532 bp.This protein was found to contain the peptide sequence.The calculated molecular mass and isoelectric point(PI)of deduced protein were 59.44 kDa and 6.83.The results of the expression showed that the mRNA of two genes were expressed in the hemocytes,hepatopancreas,muscle,gill and mantle of mussels.MMP1 was the highest expression in hemocytes and the lowest in the mantle.The expression of MMP19 was the highest in hemocytes,followed by hepatopancreas.The expression of MMP1 gene was significantly increased at 24 h in hepatopancreas and hemocytes after the stimulation of A.hydrophila.The expression levels of MMP1 in the hemocytes after the stimulation of PGN was significantly up-regulated at 3 h,then began to decreased.The expression change of MMP19 had no obvious in the hepatopancreas after stimulation with PGN and A.hydrophila,the expression level was up-regulated and was the highest at 3 h in the hemocytes after PGN stimulation.The stimulation of A.hydrophila was up-regulated in hemocytes,and the expression was the highest at 24 h.After trauma,the results showed that MMP1 genes had the highest expression level in the third day,MMP19 genes had the highest expression level in the first day,following the increase of time,the expression of two genes were decreased,and the expression level recovered to steady state at the fifteenth day.The Product of gene amplification was transferred to the expression vector of PET-28 a after BamHI,Hind III and HindIII,EcoRI double enzyme,and the prokaryotic expression plasmids of pET-28a-MMP1 and pET-28a-MMP19 were successfully builded.The plasmids were transferred into Rosetta-gami(DE3)of E.coli,SDS-PAGE analysis showed that the recombinant protein existed in inclusion body form as insoluble protein.After purificationand the protein,the protein concentration of the MMP19 was 0.094 mg/m L.