| Objectives:The protein carbonylation is an irreversible modification in oxidative stress and damage that amino acid residues is attacked from the radical transforming into an irreversible carbonylation production.Protein carbonylation is the manifestation of protein oxidative damage due to the structure and function of protein changes.In order to accurately assess the degree of protein oxidative damage and functional damage,the relation between protein oxidative damage and function is explored based on accurately assess the degress on protein damage.Methods:1.The samples of fresh milk are stored at room temperature and each group made 3parallel groups.According to temperature and different storage methods,These groups are divided,according to temperature and different storage methods,into the different groups:room temperature storage group(the sample measurement in every each 4 hours ranging from 0 to 24 hours,a total of 7 samples),room temperature vacuum group(the sample freshness measurement in every each 4 hours ranging from 0 to 24 hours,a total of 7 samples),the group with antibiotics added at room temperature(the sample measurement in every each 4 hours ranging from 0 to 24 hours,a total of 7 samples),the group with freeze-thaw samples at-20℃(There are 6 groups of 30 samples ranging from0 to 5 freeze-thaw cycles),the group with freeze-thaw cycles and vacuum at-20 ℃(There are 6 groups of 30 samples ranging from 0 to 5 freeze-thaw cycles).Each group contains 3 parallel groups.The groups at room temperature was used as a control,with the method of the protein carbonylation content chemical method,comparing with the group of vacuum and antibiotics.The groups in the-20 ℃ freeze-thaw were compared with the-20℃ vacuum group and the-20℃ normal group.The collection of fresh milk samples at different temperatures cycless of 25℃,4℃,-20℃,and-80℃.The fresh milk samples were divided into 4 freeze-thaw groups with different temperature gradients,quick-freeze and quick-thaw group,slow-freeze and quick-thaw groups,quick-freeze slow-thaw groups,slow-freeze and slow-thaw groups,freeze-thaw groups at-20℃.Each group contains 3 parallel samples.2.There are three serum groups such as the physical examination groups in 2019,the physical examination groups in 2012 and the physical examination groups with freeze-thaw oxidative damage model in 2019.Each group was selected 22 samples to determine the clinical biochemical test and protein carbonylation chemical test.The clinical biochemical test was based on the methods to determine the liver function test indicators and the glucose metabolism function.The serum biochemical data of the2019’s physical examiner,the 2012’s physical examiner,the 2019’s physical examiner using the model of oxidative damage carbonylation.The differences and commonalities between the two groups of biochemical indicators and protein carbonylation content are compared to evaluate the feasibility of model establishment.3.The establishment of the protein oxidative damage carbonylation model was using with the method of hepatitis B antibody double antigen ELISA experiment and the oxidative damage model kit ELISA enzyme-labeled antibody and antibody serum samples.There are four groups in this test,combining normal antibody serum samples with normal enzyme-labeled Antibodies,The group 1 is enzyme-labeled antibody with test samples,The group 2 is carbonylation damage enzyme-labeled antibody with test sample.The group 3 is carbonylation damage enzyme-labeled antibody with carbonylation damage test sample.The group 4 is enzyme-labeled antibody with carbonylation damage test sample.The test of groups is using the methods of protein carbonylation chemical test and ELISA kit test.The comparison of the results about ELISA and protein carbonylation content of each group was considered in the oxidative damage model and the normal test.The paired test analysis was used to analyze the comparison of the oxidative damage degree of the enzyme-labeled antibody and the serum to be tested.4.The model establishment is base on the protein oxidative damage carbonylation with measuring the PCR and RT-PCR experimental methods,using the oxidative damage kit Taq enzyme and the human DNA test samples.The groups were divided into 4 groups including Taq enzyme with human DNA test samples(group 1),carbonylation damage Taq enzyme with human DNA test samples(group 2),carbonylation damage Taq enzyme with carbonylation damage human DNA test samples(group 3),Taq enzyme with carbonylation damage human DNA test samples(group 4).The PCR qualitative experiment and RT-PCR quantitative experiment detection results were tested.The comparison of the results of each pair groups were analysize the oxidative damage model and the normal determination combination,and then analyze the oxidative damage caused by the model to test human DNA samples and Taq enzyme.Results:The test is based by 3 groups with milk at room tempture as the control group and 2test groups(placing vacuum and antibiotics method of milk).The first test about the milk samples at room temperature under vacuum and the milk samples at room temperature with antibiotics are all increased with time at 0,4,8,12,16,20 and 24 hours.Protein carbonylation content gradually increased.at 0,4,8,12,16,20 and 24 hours of milk placed at room temperature,the average protein carbonylation content was 9.58,10.14,18.28,28.09,54.38,77.84 and 127.28 μ mol/g.The increase in protein carbonylation content is due to the oxidative damage of the protein rather than the growth of bacteria reduce the quality of milk under aseptic conditions.The aseptic freshness of milk is related to protein carbonylation.The mean protein carbonylation content of antibiotic-containing milk was 8.37,12.94,14.08,22.87,32.00,73.83 and 89.78 μmol/g,at 0,4,8,12,16,20 and 24 hours,in room tempture.The mean protein carbonylation content of milk without antibiotics was 9.58,10.14,18.28,28.09,54.38,77.84 and 127.28 μmol/g.32.00 and 54.38μmol/g and 89.78 and 127.28μmol/g were observed significantly differences at 16 and 24 hours.The condition of adding antibiotics is begining from the 8th hour at room temperature that the protein carbonylation content index content is compared with ordinary pasteurized milk without antibiotics is significant differences.The protein carbonylation content of the vacuum sample is 14.47,23.39,31.63,43.63,50.33 and 52.18μmol/g,while the protein carbonylation content of the non-vacuum sample is 14.56,26.17,55.22,89.24,110.21 and 182.88 μmol/g.The results of the vacuum storage method and the results of the non-vacuum storage method have little difference in the numerical value of the 1st and 2nd freeze-thaw cycles,showing that the vacuum method has better stability than the non-vacuum method,and the last three cycles show a large numerical difference.There were significant differences in the 3rd and 4th freeze-thaw cycles,and the largest difference appeared in the 5th freeze-thaw cycle(ie 43.63 vs 89.24,50.33 vs 110.21 and 52.18 vs 182.88 μmol/g).Under different freezing conditions,ordinary freezing storage at-20℃was used as the control group.During the control process,the ANOVA analysis of variance for the slow freezing method indicated P <0.001(with statistical significance).Therefore,slow freezing under aseptic in low temperature conditions affects the carbonylation content of protein,showing that the aseptic state of slow freezing is significant differences.The carbonylation content of protein increases with the number of freeze-thaw cycles regarded as an experimental model of protein carbonylation.2.There are three groups have undergone physical examination and the model to test about the 2012’s physical examination and the 2019’s physical examination.Independent sample t-test is used for statistical analysis.The fresh serum sample and the serum of the control group were not subjected to repeated.Freeze-thaw model were tested for protein carbonylation content.The freeze-thaw cycle analysis was carried out through the protein carbonylation oxidative damage mechanism.11 samples in each group were repeatedly measured and the average value was taken to determine the protein carbonylation content.Fresh serum samples were used as the control group,and the freeze-thaw samples after the freeze-thaw cycle were the experimental group.The mean of the experimental group and the control group were 2.545 and 1.965 μ mol/g,indicating that the protein carbonylation content after the freeze-thaw cycle increased compared with the carbonylation protein content of fresh serum samples to be tested.According to statistical analysis,there is a significant difference in the protein carbonylation content between the freeze-thaw group of the test sample and the healthy control group(P<0.001).The protein carbonylation content of the patients with repeated freeze-thaw physical examination is significantly higher than that of the healthy control group.difference.The carbonylation content of serum samples to be tested in 2012 and 2019 were 2.551 and 1.450 μmol/g after multiple measurements and the average value.The content of carbonylated protein in old serum in 2012 was more than that in 2019.The comparison of the protein carbonylation content of the test samples was significantly different between groups(P<0.001).The protein carbonylation content of the physical examination in 2012 was significantly higher than that of the healthy control.It can be considered that the serum protein carbonylation oxidative damage of the physical examination in 2012 was higher than that in 2019 Control group of medical examiners.Protein carbonylation content,alanine aminotransferase,total protein,total bilirubin,high-density lipoprotein,alkaline phosphatase have the same difference P<0.05 is statistically significant,indicating that the biochemical indicators of the freeze-thaw experiment have changed It is consistent with the changes in the biochemical indicators of the old serum experiment.Among the14 biochemical test items,there are 4 biochemical items that are not different,and the coincidence rate is 100%;there are 10 biochemical indicators that all have differences.There are 6 items that are completely coincident,the coincidence rate is 60%,and the difference rate data of all differences and no differences shows that the average coincidence rate is 80%.Provide data support for the detection of biochemical indicators for oxidative damage damage models.The establishment of the oxidative carbonylation damage by the test groups have been divided into 4 groups.Each group has been hold 40 samples,based on the antibodies and enzyme label.In the pair group judging the freeze-thaw antibodies and normal samples,the value of means were 0.822 and 0.581.P<0.05(0.040),there is a statistical difference,and the two have certain differences in paired sample t-test analysis.The other pair group is compare between freeze-thaw kit enzyme label and the normal kit enzyme label.Each group has 40 samples,a total of 80 cases,the means are0.902(freeze-thaw)and 0.501(normal),and P<0.001.There is a statistical difference,indicating that the two have certain differences.Two factors about freeze-thaw can be combined 6 comparsions,comparsion 1 about ordinary serum + ordinary enzyme label and ordinary serum +freeze-thaw enzyme label;ordinary serum + ordinary enzyme label and freeze-thaw serum + ordinary enzyme;common serum + enzyme label and repeated freeze-thaw serum + freeze-thaw enzyme label;freeze-thaw serum + common enzyme label conjugate and common serum + freeze-thaw enzyme label;freeze-thaw serum +common enzyme label and freeze-thaw serum + freeze-thaw enzyme label;common serum + freeze-thaw enzyme label and freeze-thaw serum + freeze-thaw enzyme label.P<0.05(0.05)was statistically significant,with a 95% confidence interval,using the paired variance t test.Among them,the combination of normal enzyme label conjugate +normal serum antibody group and freeze-thaw enzyme label + normal serum antibody group are significantly different,P<0.001(0.000)is statistically significant,with 99 %Confidence interval.4.The establishment of the oxidative damage model is based on the feasibility of the experiment judged from the perspective of quantitative experiments.In order to explore the impact of protein carbonylation on the Taq enzyme in the fluorescent real-time quantitative PCR kit based on the establishment of the oxidative damage model judging from the perspective of quantitative,a single-factor independent sample t-test analysis is used.There are two groups including normal Taq enzyme group and the Taq enzyme group with the modification of each groups.There are 12 samples in each group and 24 cases in total.The means of the two groups are 0.745 and 0.075 respectively,and P<0.001 is statistically significant.The determination result of protein carbonylation is caused by the human genomic DNA samples in the fluorescence real-time quantitative PCR measurement,the single factor independent sample t test is frozen through the protein carbonylation model thawing divided into groups including 12 samples in each of the two groups about 24 cases,the means were 0.565 and 0.265,and the P <0.001,was statistically significant.The result shows that the human genomic DNA extract after freezing and thawing through the protein carbonylation model may be oxidized,which will affect the experimental results.Conclusion:1.Aseptic freshness is related to protein carbonylation.The degree of aseptic freshness can be damaged by measuring protein carbonylation content.The vacuum state can maintain the aseptic freshness state to a certain extent.The increase in the cycles of freeze-thaw accelerates the reduction of aseptic freshness,and gradually reduces the hazard of cryopreservation(slow freezing)to samples,and the freezing method has a certain impact on aseptic freshness.2.Protein repeated freeze-thaw technology is based on apply chemical method to determine protein carbonylation technology to detect protein oxidative damage ability.The establishment of freeze-thaw carbonylation model shows that the experimental establishment of protein carbonylation model is successful.The model shows that carbonylation has a biochemical detection of blood lipids.The indicators and some enzyme functional biochemical testing indicators have an impact,and little impact on the glucose metabolism testing indicators and kidney biochemical testing indicators.3.The protein carbonylation damage ability of the antibody and standard protease of the examiner was tested.Protein carbonylation has a small impact on the immune function of the body at the antibody level,and has a greater impact on the protein damage of standard protein enzymes.4.Carbonylation has a significant impact on the Taq enzyme in PCR and RT-PCR experiments,the oxidatively damages the DNA extract,and then the experimental results have a certain impact.The mechanism of DNA damage should be further explored. |
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