Expression Of Matrix Metalloproteinase-12 In Mitral Valve With Rheumatic Heart Disease

Matrix metalloproteinase-12 (MMP-12), also known as human macrophage elastase(HME), belongs to a family of zinc-dependent endopeptidases. MMP-12 is involved in many physiological processes such as the vascular remodeling, conversion of plasminogen into angiostatin, development of airway inflammation and remodeling. MMP-12 was also involved in the remodeling of extracellular matrix in many diseases, such as abdominal aortic aneurysm, atherosclerosis, emphysema, cancer, rheumatoid arthritis, liver and lung fibrosis. In this work, we will determine the expression of MMP-12 in mitral valve with rheumatic heart disease.First, the protease was expressed in E. coli. The full-length gene (1.1 kb, GenBank Accession No.:AAA58658.1) encoding MMP-12 (45 kDa) was amplified from a human hepatoma cell line cDNA library using the polymerase chain reaction (PCR) method and comfirmed by sequencing. The PCR products were restricted with BamH I and Xhol, and ligated into BamH I and Xhol restricted sites of the pET-X17b vector (Novagen Inc.) without any tags at the terminuses. The recombinant plasmid was transformed into E. coli strain BL21 (DE3) and the transformants were selected on LB agar plates containing 100μg/ml Ampicillin. Positive clones with an insert of the right size were confirmed by DNA sequencing. Cells were cultured in LB medium and induced by IPTG. The recombinant protease was expressed in the form of inclusion body. The inclusion body was dissolved in 5M guanidine hydrochloride and then diluted in refolding buffer at 4℃for 40h. The diluent was concentrated by ultrafiltration. Enzymatic assays of the protein were carried out using EDANS/DabcylPlusTM FRET peptide as the substrate, with its fluorescence being monitored at Ex/Em=355/460 nm upon proteolytic cleavage, digestion assay and zymography. Second, the expression of MMP-12 was determined in rheumatic mitral valve, taking non-RHD mitral valves as the control. The expression of MMP-12 has been detected by different methods, including reverse transcription polymerase chain reaction (RT-PCR) Western Blot, immuno-histochemistry and fluorescence resonance energy transfer(FRET). Meanwhile, the activity of MMP-12 in serum was also detected by fluorescence resonance energy transfer.In summary, the novelty of the research is embodied as follows:1) The expression vector of pET-17xb-MMP12 was successfully constructed, and the inclusion body of expressed MMP-12 catalytic domain was denatured and refolded.2) The activity of MMP-12 was detected with fluorescence and the reaction condition was optimized. 3) The expression of MMP-12 in mitral valve with rheumatic heart disease (RHD) has been verified on gene, protein and tissue level, providing a fundamental basis for further exploration of MMP-12 in the remodeling mechanism of extracellular matrix (ECM) of mitral valve with rheumatic heart disease.