| Superoxide Dismutase(SOD)is a kind of protein with antioxidant activity that exists widely in nature and can specifically remove Superoxide produced in the process of metabolism.Because of its important biological function,SOD has a wide application prospect in food additive,agricultural production,cosmetics,bio-medicine and other fields.However,the defects of natural SOD,such as poor stability,low cell affinity and easy degradation by protease,greatly restrict its development and application.In recent years,with the construction of a large number of enzyme mutants and the determination of the corresponding thermal stability data,it is possible to improve the enzyme stability by using computer aided software.In the study of the relationship between protein structure and function,it is necessary to design the amino acid sequence rationally and then verify it by experiment.It has been reported that amino acid virtual mutation based on thermal stability is used to evaluate the effect of mutation on protein stability,and mutation energy(ΔΔGmut)is used to determine amino acid site combinations.Then,molecular dynamics simulation(MD)analysis is used to determine the optimal simulation sites or combinations to obtain candidate mutants.Finally,the mutants with better thermal stability and acid and alkali resistance are obtained through experimental verification.About 10%of people in the world suffer from gastric Ulcer disorder(GU).The dynamic balance break of oxidation-antioxidant system is one of the important causes of GU.Excessive alcohol consumption is the most common cause of gastric ulcers.Traditional gastric ulcer treatment methods are mainly to inhibit the damaging factors and restore the natural structure of gastric mucosa,such as the use of antisecretory drugs-proton pump inhibitors and H2 receptor blockers,etc.,effective but accompanied by obvious side effects.Therefore,it is urgent to develop new methods to treat gastric ulcer with effective anti-inflammatory and antioxidant stress.In this paper,MnSOD derived from Thermus Thermophiles HB27 was used as th e research object,amino acid optimization was carried out by site-directed mutation technology,and high stability MnSOD mutant strains were constructed and screened.Meanwhile,the highly stable manganese superoxide dismutase mutant was used to explore its effect on acute gastric ulcer model mice and its mechanism.The main research contents and results are as follows:1.Since the MnSOD gene derived from Thermus Thermophiles HB27 was used as the template,four single mutants of MnSOD were constructed by seamless cloning method through PremPS online platform prediction,multiple sequence comparison and other bioinformatics methods.On this basis,three double mutant escherichia coli expression vectors(pET28b-MnSOD-L117F/K138V、pET28b-MnSOD-L121 F/K138V、pET28b-MnSOD-K138V/A182P)were constructed.By measuring the activity of recombinant protease,thermal stability,acid resistance,stability in artificial simulated gastric and intestinal fluid,and coagulation,the mutant with high stability was screened out.The results showed that the enzyme activity of L121F/K138V mutant was 15.66%higher than that of wild type(wt),other mutants showed different degree of specific viability decline,K138V/A182P decreased by about 55.57%compared with wild type.The constructed double mutant still had excellent broad-spectrum thermal stability,as the residual enzyme activity remained above 90%after incubation at RT to 90℃ for 1 h.Besides,the residual enzyme activity of L117F/K138V mutant was 94.81%after incubation at 90℃ for 24 h,which was significantly higher than 73.94%of wt-MnSOD.And They had good acid resistance,as their enzyme activity were more than 60%in the range of pH3-6.In addition,after 12 h of pepsin enzymatic hydrolysis,the residual enzyme activity was more than 70%,and the residual enzyme activity of trypsin enzymatic hydrolysis was more than 95%after 12 h.,The results of high throughput multi-parameter protein stability analyzer further confirmed that the particle size distribution of L117F/K138V mutant was only 389.36 nm at 95℃,which was much smaller than that of other mutants(1000 nm),indicating that it was only partially denatured and precipitated at high temperature.And the thermal stability and protein aggregation of L117F/K138V(Tm=88.7℃,Tagg=75.07℃)and the K138V(Tm=88.71℃,Tagg=76.82℃)mutant was improved compared with the wild type(Tm=86.95℃,Tagg=72℃).As a conclusion,the protein stability of the mutant constructed by laboratory molecular modification were improved2.At the same time,homologous modeling was used to analyze the mutation sites using biological information to analyze the mechanism of stability change.In the secondary structure,L117,L121 and A182 residues participate in a helix,while K138 residues constitute β fold.K138V mutation changes the hydrophobicity of local protein conformation compared with other mutations.Meanwhile,the L117F and K138V site mutations affect the 12th α helix,extending one amino acid residue length toward the C-terminal,which increase protein rigidity and stability.In addition,point mutation can also affect protein stability through changes in protein hydrogen bond network and active center.The directed mutations at sites 117 and 138 had positive effects on the stability of MnSOD protein,K138V and the L117F/K138V mutant constructed in this study showed outstanding stability.3.We established acute gastric ulcer model in KM mice by gavaging the mixture of ethanol and HCl in one time,and used this model to evaluate the alleviating effect of MnSOD-L117F/K138 mutant on ethanol/HCl-induced gastric ulcer and its mechanism.MnSOD(2000 IU/10 g B.W.)was gavaged continuously for 14 days.On the 14th day,acute gastric ulcer model was constructed by gavage of gastric ulcer inducer to explore the protective and preventive effects of MnSOD on GU mice.After the occurrence of ulcer,SOD was administered for 5 days to explore the therapeutic effect of SOD in the early stage of ulcer.Ethanol/HCl mixture can cause severe gastric ulcer,abnormal gastric edema,extensive hemorrhagic necrosis and typical ulcer lesions,while MnSOD can significantly reduce its severity.The mechanism of action was analyzed from the perspectives of daily activity index,non-specific immunity,histopathology and expression of GU related genes.Through comparative analysis,we found that MnSOD itself had no damage to gastric mucosa,and could promote the growth of mice,as the body weight increased about 8.04%compared with normal saline control group.MnSOD could increase thymus and spleen index and enhanced non-specific immunity.Among the preventive effects of MnSOD on GU,the inhibition rate of gastric ulcer was 53%.After 5 days of treatment with MnSOD,the ulcer inhibition rate was 69%,which was better than omeprazole(0.2 mg/10 g b.w.),with ulcer inhibition rate of 32%.And the total inflammation index was significantly reduced.In the process of GU prevention and treatment,MnSOD could well regulate the levels of inflammatory cytokines(TNF-α,IL-1β,IL-6,IL-10),promote the expression of prostatin E2,and significantly up-regulate the expression of Nrf2(P<0.01),a key antioxidant transcription factor.These results suggest that MnSOD can play a good role in the prevention and treatment of GU,significantly increase the level of Nrf2 in gastric tissue,effectively activate the antioxidant stress pathway to remove ROS,and improve oxidative stress.It indirectly regulates inflammatory mediators and plays the role of mucosal barrier to promote gastric mucosal injury repair.The above results indicate that MnSOD can be modified by site-directed mutation to improve its stability.Moreover,MnSOD has considerable preventive and adjuvant therapeutic effects on ethanol/HCl type acute gastric ulcer in clinical practice.This study provides a reference for the modification of protein by genetic engineering and the widening of the application value of MnSOD. |
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