舌苔上清液中蛋白提取方法的建立及优化

Establishment and Optimization of Extraction Methods for Protein in Supernatants of Tongue Coating

目的:建立舌苔上清液中蛋白的提取及优化方法。方法:采用不同的离心条件、不同过滤器孔径及不同样本溶剂或芯片的选取等方法,对30例正常人、30例慢性肾衰竭(CRF)患者舌苔样本进行分组处理。对舌苔上清液采用紫外分光光度计检测各组样本的OD值,并计算各组样本的蛋白浓度;通过SPSS11.5软件统计分析,筛选出蛋白浓度相对稳定的处理条件;基于SELD I-TOF-MS技术,使用不同类型的蛋白芯片点样,根据图谱中蛋白波谱峰分布情况,确定最为优化的样本处理条件。结果:在选用不锈钢金属小匙取样溶于0.9%生理盐水,经过1 500 r/m in,离心2 m in的条件后取上清液,NP20蛋白芯片点样结果显示的蛋白波峰数最多,波峰相对高度最高且最为稳定。结论:该实验为舌苔蛋白质组学的理论研究提供一定的科研思路与方法。

Objective： To establish and optimize the methods for extracting the protein from supernatants of tongue coating.Methods： The tongue coating samples from 30 cases of healthy people and 30 cases of chronic renal failure（CRF） were processed with different centrifugation conditions,filter pore size,sample solvents or chips.The OD values of samples from all groups were detected by ultraviolet spectrophotometric assay and the protein concentration were calculated.With SPSS11.5 software analysis,the processing condition for stable protein concentration was identified.Then mapping the graphs of protein concentration by calculating the concentration of protein of all group samples,and ultimately obtaining the treatment approaches,which makes protein concentration relatively stable by comparison and screening.Based on SELDI-TOF-MS technology,different types of protein chips were utilized to determine the optimal conditions for sample processing according to the distribution of the protein spectral peaks.Results： When the samples were collected with metal spoon,dissolved in 0.9% salt-water and centrifuged at 1 500r/min for 2 min to acquire the supernatants,the NP20 protein chip showed that the protein peaks had the largest numbers and maximal height and had been the most stable.Conclusion： This research can provide some ideas and methodology for the theoretical study of tongue coating proteomics.