Preparation Of Recombinant PTD-Cu/Zn SOD And Examination The Protection Of The Recombinant Protein On Myocardial Injury

Myocardial ischemic-reperfusion injury, main myocardial injury during the operations of cardiac surgery especially in the coronary artery bypass graft, is closely related with oxygen-free radicals overproduction in heart at the early stage of reperfusion. Removing the oxygen-free radicals in cardiomyocytes may attenuate the cardiac reperfusion injury. Studies have indicated that superoxide dismutases (SOD) are the eukaryotic enzymes known to remove toxic superoxide anions and protect the heart by blood-borne elements.Cu,Zn superoxide dismutase (Cu/Zn-SOD, SOD) is a highly conserved in eucaryon cytoplasm as well as peroxisome, and ubiquitously expressed anti-oxidant enzyme involved in the conversion of toxic superoxide anions into molecular oxygen and hydrogen peroxide. Studies showed that Cu/Zn SOD had the ability of clearing the oxygen-free radicals like lipid peroxidation and superoxide anions, preventing cerebral ischemia/reperfusion (I/R) injury. Cu/Zn SOD could have a beneficial effect of afterload reduction on oxidative stress in heart failure and constitute an important defense element for the hearts from ischemic-reperfusion injury. Also it has been indicated that susceptibilities of hepatocytes and endothelial cells to H2O2-induced injury are altered by changes in the intracellular activity of CuZn-SOD and myocytes coping with chronic oxidative stress results from imbalance between intracellular oxygen radical-generating and scavenging systems. However, Cu/Zn-SOD has not any special receptor or channel in cell membrane, and ectogenic Cu/Zn-SOD is limited to be transferred into cells and tissues to play clinical effects.Protein transduction domain (PTD), consisting of11-34amino acid residues, serves as trans-elements that facilitate membrane penetration by heterogeneous proteins and oligonucleotides. Therefore, the major role of PTDs in these phenomena is transferring large proteins highly into cells to play great biological effect. This study is to identify plasmid with human PTD and Cu/Zn SOD being fused, which can efficiently cross cell membrane, and then plasmid can be produced in Escherichia coli; further the fusion protein of PTD-Cu/Zn SOD is extracted. Finally this PTD-Cu/Zn SOD protein is used to assess its property of intercellular transport, anti-oxidation and protection on cultured cardiomyocyte under hypoxia reoxygenation injury (HRI) and preventing myocardial ischemia/reperfusion injury induced by ligation of LAD.PART ONE Construction, expression and purification of recombinant PTD-Cu/Zn SODObjective To constructe, expresse and purify the recombinant PTD-Cu/Zn SODMethods According to characterize of PTD, trans-membrane activity was designed and the coding sequence (CDs) primer for full-length of human Cu/Zn SOD was amplified, then after these, PET16b prokaryotic expression vector as well as pGEM(?)-T cloning vector was selected. The total RNAs abstracted from embryo liver were as templates used to reverse transcribe and amplify the CDs for full-length Cu/Zn-SOD, and the full-length CDs of Cu/Zn-SOD produced after these was purified by Adenine, inserted into pGEM-T vector and changed into the pGEM-Cu/Zn-SOD (CDs) vector. The linear vector with double cohesive ends and full-length Cu/Zn-SOD CDs elements were recycled and inserted directionally into pET-16b vectors to become to the pET-Cu/Zn-SOD (CDs) prokaryotic expression vectors. At last these small segments of double-stranded DNA sequences were inserted into the above linear vectors, and became pET-PTD-Cu/Zn-SOD(CDs) prokaryotic expression vectors. These vectors were inserted into competence E. coli BL21(DE3) with pET-Cu/Zn-SOD, then the protein were collected.ResultsThe sequence of amplified PTD was consistent with the designed PTD. The PTD-Cu/Zn SOD fusion gene was amplified successfully and the length of these quence was567bp.The PTD-Cu/Zn SOD (CDs) prokaryotic expression vectors inplasmid were successfully constructed and inserted into E. coli BL21to produce alarge quantity of recombinant PTD-Cu/Zn SOD proteins.The expressed fusion proteinPTD-Cu/Zn SOD was souble and about20kDa which was accroding with the expectedrecombinant products.ConclusionsWe successfully constructed the PTD-Cu/Zn SOD plasmid to express the recombinant protein PTD-Cu/Zn SOD.The expression and purification of the fusion protein will facilitate further functional study and clinical application of protein transduction technology. PART TWO Protection of PTD-Cu/Zn SOD on myocardial injury induced by hypoxia reoxygenationObjective To examine the penetrability of PTD-Cu/Zn SOD into cultured cardiomyocytes and the protection of PTD-Cu/Zn SOD on myocardial injury induced by hypoxia reoxygenationMethods The activity of protein recombinant enzyme of PTD-Cu/Zn SOD was exmined with pyrogallol oxidation rate.The transmembrane ability of fusino protein was investigated by examination of PTD-Cu/Zn SOD in cells using IHC, Western blot and RT-PCR resepectively on cultured cardiomyocytes. HRI was achieved by exposing card iomyocytes to12h hypoxia followed by2h reoxygenation. The anti-oxidant effect of PTD-Cu/Zn SOD in cardiomyocyte induced by hypoxia-reoxygenation injury was tested by analysing the mitochondria membrane potential, the level of superoxide anion. The protection of PTD-Cu/Zn SOD on cardiomyocyte indury induced by hypoxia-reoxygenation was investigated by examintion of cardiomyocytes apoptosis.Results The results showed that PTD-Cu/Zn SOD maintained the antioxidant activity of SOD and still kept the PTD vectors transmembrane ability. PTD-Cu/Zn SOD suppressed the production of superoxide anion and maintained mitochondrial membrane potential in cardiomyocytes during hypoxia and reoxygenation. PTD-Cu/Zn SOD decreased apoptosis of cardiomyocytes induced by hypoxia-reoxygenation injury.Conclusion PTD-Cu/Zn SOD could effectively penetrate cell membrane, scavenge intracellular free superoxide anion, protect mitochondria from damages, and attenuate the hypoxia-reoxygenation injury in cultured cardiomyocytes. PART THREE Protection of PTD-Cu/Zn SOD on myocardial ischemia reperfusion injuryObjective To examine the protection of PTD-Cu/Zn SOD on myocardial injury induced by IRI.Methods Myocardial ischemia reperfusion injury was induced by ligaturing for30minutes and then reperfusion for120minutes of left anterior descending of coronary artery. PTD-Cu/Zn SOD or Cu/Zn SOD were treated from left atrium post reperfusion. The area of myocardial infarction was examined by TTC staing. The transmembrane ability of fusino protein was investigated by examintion PTD-Cu/Zn SOD in cells using IHC, Western blot and RT-PCR resepectively. CK, CK-MB, LDH, MDA and apoptosis with necrosis of myocardia were examined at120minutes post-reperfusion.Results Immunofluorescence analyses indicated that PTD-Cu/Zn SOD was in the cytoplasm of most cardiac muscle, and quantitative analysis showed that the SOD in the cardiac myocyte was from exogenous PTD-Cu/Zn SOD through the cell membrane. PTD-Cu/Zn SOD decreased the area of myocardial infarction, the levels of CK, CK-MB, LDH, MDA, myocardial apoptosis and necrosis induced by myocardial ischemia reperfusion injury.Conclusion PTD-Cu/Zn SOD could effectively penetrate cell membrane scavenge intracellular-free superoxide anion, prevent myocardial infarction, and attenuate the myocardial apoptosis and necrosis induced by myocardial ischemia reperfusion injury.