| 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,tissue inhibitor of metalloproteinase TIMP1,2 cDNA were cloned from H.cumingii using rapid amplification of cDNA ends.The expression of TIMP12 mRNA was measured by real-time fluorescence quantitative PCR in the five organizations in a health mussel such as gills,mantle,cranial muscle,hemolymph,hepatopancreas,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 TIMP1 cDNA was 1160 bp in length and contained a 5' untranslated region(UTR)of 40 bp,a 3' UTR of 412 bp and 708 bp open reading frame(ORF)encoding a total of 235 amino acid residues.Analysis of SignalP 4.0 by Expasy online site found the molecular weight of the predicted TIMP1 was 27.26 kDa,with the calculated PI being 8.89,the total number of negatively charged residues(Asp + Glu)of 26,the total number of positively charged residues(Arg + Lys)35.Signal P program analysis showed that it had signal peptide.The length cDNA sequence of the cloned TIMP2 was 729 bp and contained a 453 bp open reading frame(ORF)encoding 150 amino acid residues and had 38 bp of 5 ' untranslated region(UTR)and a 3' UTR of 238 bp.This protein was found to contain the peptide sequence.The calculated molecular mass and isoelectric point(PI)of deduced protein were 16.58 kDa and 8.72.The total number of negatively charged residues(Asp + Glu)was 11,and the total number of positively charged residues(Arg + Lys)was 15.The results of the expression showed that the m RNA of twogenes were expressed in the hemocytes,hepatopancreas,muscle,gill and mantle of mussels.TIMP1 was the highest expression in hemocytes and the lowest in the mantle.The expression of TIMP2 was the highest in muscle,followed by hemocytes.The expression of TIMP1 gene was significantly increased at 24 h but it had no significant differences in hepatopancreas after the stimulation of Aeromonas hydrophila.The expression levels of TIMP1 in the hemocytes after the stimulation of PGN was significantly up-regulated at 6 h,then began to decreased.The expression change of TIMP2 had no obvious in the hepatopancreas after stimulation with PGN and A.hydrophila,the expression level was up-regulated and was the highest at 48 h in the hemocytes after PGN stimulation.The stimulation of A.hydrophila was up-regulated in hemocytes,and the expression was the highest at 3 h.After trauma,the results showed that TIMP1,2 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-32 a after HindIII and EcoRI double enzyme,and the prokaryotic expression plasmids of pET-32a-TIMP1 and pET-32a-TIMP2 were successfully builded.The plasmids were transferred into Rosetta-gami(DE3)of E.coli,The results showed that the recombinant protein existed in the precipitation,as insoluble protein.After purificationand the protein,the protein concentration of the TIMP1 was 0.140mg/mL and the protein concentration of the TIMP2 was 0.310 mg/m L,it was purified by reverse zymography found the recombinant protein of TIMP1,2 could inhibited the activity of MMP2. |
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