High Expression Of RbCu,Zn-SOD In Pichia Pastoris And Its Protective Effect On Lead Poisoning Mice

Oxidative stress（OS）refers to the imbalance between oxidation and anti-oxidation in the body,resulting in a large amount of reactive oxygen species（ROS）being generated and causing cell damage.ROSs include superoxide anion(O^2-),hydrogen peroxide（H₂O₂）,hydroxyl radical（·OH）,and nitric oxide（NO）,etc.Excessive ROSs will destroy important macromolecules such as nucleic acids and proteins,resulting in lipid peroxidation damage of biological membranes,and initiate and develop carcinogenic processes.Lead pollution is a common problem in the modern environment.Lead poisoning can cause oxidative stress reaction in the body,and serious damage to cardiovascular system,nervous system and immune system.For the treatment and prevention of mild lead poisoning,the use of antioxidant substances to participate in the body’s redox reaction,eliminate excess free radicals and other active metabolites and improve the body’s immunity at the same time,it can eliminate the harm that the metal ion chelating drugs to expel lead and beneficial metal elements.Superoxide dismutase（SOD）is an endogenous antioxidant enzyme ubiquitously in nature and is the most powerful antioxidant in cells.SOD widely exists in animals,plants and microorganisms.Extracting SOD from animal blood has the risk of pathogen infection,and the process of extracting SOD from plants and microorganisms is complicated,with extremely low yield and a waste of human and financial resources.As a fast,economical,efficient and safe foreign protein expression system,the Pichia pastoris expression system has been widely studied and applied to obtain recombinant SOD,and satisfactory results have been achieved.In this study,two Pichia pastoris expression systems,p GAPZαMUT-Cu,Zn-SOD and p PICZα-Cu,Zn-SOD,were constructed to express rb Cu,Zn-SOD.p PIC-SOD was screened as an engineering strain for optimization of expression conditions,pilot fermentation culture and purification of target protein.The enzymological properties of the obtained target protein were studied,and the effects of recombinant SOD on organ coefficient,blood and tissue lead concentration,oxidative damage and liver and kidney histopathology of lead poisoning mice were also explored.The specific research contents are as follows:（1）The recombinant plasmids pGAPZαMUT-Cu,Zn-SOD and p PICZα-Cu,Zn-SOD were constructed by modifying the expression vector and optimizing the whole gene sequence of bovine Cu,Zn-SOD was optimized according to the codon usage preference of Pichia pastoris.The results of double enzyme digestion electrophoresis showed that there was a characteristic band at 487bp,which was consistent with the expectation.which was in line with expectations.The linearized recombinant plasmids were electroporated into Pichia pastoris competent cells.Zeocin resistance was used to screen p GAP-SOD and p PIC-SOD positive transformants,and the SDS-PAGE results of shake flask cultured protein products showed that there was a characteristic band at 17k Da,which was consistent with the expectation.The p GAP-SOD and p PIC-SOD were purified by metal affinity chromatography,the enzyme activities were determined by Marklund method and the protein concentrations were estimated by BCA method.The results showed that the enzyme activity of p PIC-SOD was 423.8U/m L,and the protein concentration was 96.3mg/L.The enzyme activity of p GAP-SOD was 295.2U/m L,and the protein concentration was 70.2mg/L.The p PIC-SOD was comprehensively selected as the follow-up experimental engineering strain.The purified protein was subjected to denaturation SDS-PAGE,and the recombinant SOD dimers were all reduced to monomers,indicating that the rb Cu,Zn-SOD engineering strain have been successfully constructed.（2）According to the results of single-factor experiments,using the Box-Behnken experimental design principle,the carbon source concentration,inducer concentration,p H and temperature were used as the factors,and the SOD activity was used as the response value to conduct a response surface optimization experiment on the fermentation conditions.The scoring order of the investigated factors to the response value is carbon source concentration>inducer concentration>pH>temperature.According to the actual operation,the culture conditions were determined as follows:the concentration of carbon source（glucose）was 2.6%,the concentration of inducer（methanol）was 1.3%,the p H was 6.2,and the temperature was 28°C,the maximum enzyme activity of recombinant SOD was 638.7U/m L,which was 1.5 times of that before optimization.（3）The engineering bacteria were fermented in a 50L fermenter.Based on the optimization results of expression conditions,a two-stage fermentation method was adopted.In the initial growth stage of the cells,the glucose solution was fed for 6hours to make the cells grow in large quantities.In the stage of protein induction and expression,fed methanol solution to induce expression for 72h to end the fermentation.The supernatant was collected by centrifuge and purified by ultrafiltration,affinity chromatography,dialysis,desalting and concentration.The results showed that the enzyme activity of the fermentation supernatant was2427.1U/m L,and the target protein concentration was 473.6mg/L.SDS-PAGE of the purified protein showed that the bands at 34k Da and 17k Da were consistent with the molecular weight of the target protein,and the sample was electrophoretic pure.The results of enzymatic properties showed that the enzyme activity of recombinant SOD was stable in the range of 40～60℃,and the enzyme activity decreased significantly when the temperature was higher than 70℃.The enzyme activity of recombinant SOD was stable in the range of p H4.0～10.0,and had good acid-base tolerance.Recombinant SOD is very sensitive to low concentration of H₂O₂,has good tolerance to low concentration of SDS and has special properties to chloroform-ethanol.（4）Animal experiments were conducted to study the effect of recombinant SOD on lead poisoning mice.Lead poisoning was modeled in mice by intraperitoneal injection of lead acetate solution.Different doses of recombinant SOD were administered for 4 consecutive weeks,and myricetin was used as a positive control group.The results showed that compared with the model group,the high-dose group and the positive control group significantly improved（P<0.05）the body weight and organ coefficient,the lead concentration in blood and liver and kidney tissue,and the oxidation stress related indicators MDA,SOD and GSH-Px of plasma,liver and kidney tissue homogenate.The middle and low dose groups also improved the oxidative damage in lead poisoning mice to varying degrees.The observation results of pathological tissue sections showed that the high and medium dose groups and the positive control group could significantly improve the pathological phenomena such as liver tissue cell necrosis and disorder,nuclear enlargement and central venous congestion,as well as glomerular atrophy and blurred kidney tissue structure caused by lead poisoning in mice.In conclusion,this study successfully constructed rb Cu,Zn-SOD in Pichia pastoris expression.Through the optimization of the expression conditions of the engineered bacteria,the production of the target protein was improved by purification and detection of the target protein.The related enzymatic properties of recombinant SOD and its protective effect on oxidative stress in lead poisoning mice were also explored,laying a foundation for the subsequent exogenous expression and theoretical research of recombinant SOD.