| Objective:To research the best reaction conditions of the complex which is combined by Nano-silver and epidermal growth factor(EGF), and to research the binding rate of the complex which is combined by EGF that is in different concentrations and Nano-silver that is in the same concentration,and to provide the basis for preparation of the complex.Methods and Material:To prepare EGF that is 200μg/ml and Nano-silver that is 5000ppm.To make up the complex in different reaction conditions.Experiment is divided into pure epidermal growth factor group,pure nano-silver group,Nano silver-epidermal growth factor complex group(referred to as complex group) and normal saline control group.The complex group is divided into three subgroups according to the different reaction conditions which is PH of solution,reacting temperature and reacting time.The first subgroup is divided into four subgroups according to PH of solution.The PH of solution is separately 5.0,6.0,7.0 and 8.0.The second subgroup is divided into three subgroups according to the reacting temperature.The reacting temperature is separately 4℃,25℃and 37℃.The third subgroup is divided into three subgroups according to the reacting time.The reacting time is separately 12hours,24hours and 36hours.Low-temperature high-speed centrifugate (4℃,13000rpm) the complex which is producted in the above different reaction conditions.The time of every centrifugation is 30 minutes.We will get the supernatant fluid after centrifugating.Centrifugate again and again until the supernatant is clear.The next step is Tricine-SDS-polyacrylamide gel electrophoresis.After electrophoresis, quantitatively analyse the protein band which is the result of electrophorsis by Gel Imaging System.We can get the binding rate of the complex which is producted in different reaction conditions.Then we will know the best reaction conditons.We will change the consistency of EGF and product the complex in the best reaction conditions.The consistency of EGF is separately 50μg/ml,75μg/ml,100μg/ml and 150μg/ml.And then processing electrophoresis and analyzing quantitatively,we will know the binding rate of complex which is in different consistency of EGF and the same consistency of Nano-silver. The changing discipline of binding rate of complex with the consistency of EGF changing will be found.Results:The epidermal growth factor group band and the complex group band can be seen.We cann't see any bands in Nano-silver group and the normal saline control group.In this experimentation,when pH of solution is 5.0,6.0,7.0 and 8.0,the binding rate is separately 17.39%±0.11%,16.09%±0.17%,28.32%±0.10%and 19.36%±0.23%,the statistical analysis showed that there was significant difference in the four subgroups(P=0.016),and there was significant difference in any two subgroups(P＜0.05),the binding rate of the complex is the highest when the pH of solution is 7.0;when the reacting temperature is 4℃,25℃and 37℃,the binding rate is separately 23.20%±1.17%,15.51%±0.74%and 29.89%±0.80%,the statistical analysis showed that there was significant difference in the three subgroups(P=0.027),and there was significant difference in any two subgroups(P＜0.05),the binding rate of the complex is the highest when the reacting temperature is 37℃;when the reacting time is 12hours,24hours and 36hours,the binding rate is separately 28.55%±0.15%,33.41%±0.24%and 17.29%±0.19%,the statistical analysis showed that there was significant difference in the three subgroups(P=0.027),and there was significant difference in any two subgroups(P＜0.05),the binding rate of the complex is the highest when the reacting time is 24hours.When the concentration of Nano-silver is 500ppm,the concentration of EGF is separately 50μg/ml,75μg/ml, 100μg/ml and 150μg/ml,the binding rate is separately 26.27%±0.58%, 21.29%±0.47%,19.03%±0.59%and 16.48%±0.24%,the statistical analysis showed that there was significant difference in the different subgroups(P=0.019),and there was significant difference in any two subgroups(P＜0.05),the binding rate of the complex is decreasing when the concentration of EGF is increasing,but the binding capacity of complex is increasing.Conclusion:We find it is the best reacting conditions when pH of solution is 7.0,when the reacting temperature is 37℃and when the reacting time is 24hours.Another discovery is that the binding rate of Nano silver-epidermal growth factor complex is gradually decreasing as the concentration of epidermal growth factor increasing. |
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