【摘要】 目的 建立人胎盘叶酸受体蛋白的提取及纯化方法,并对其进行定性定量检测。方法 采集医院分娩的正常人的胎盘样品。将胎盘剪碎,组织匀浆,酸化及中和,过柱提纯,组分鉴定,除杂并用分子筛进一步纯化。利用Bradford检测蛋白浓度,用SDS-聚丙烯酰胺凝胶电泳和银染方法进行蛋白分子量检测,用Western Blot对提取蛋白进行定性鉴定。结果 用于本实验的胎盘湿重370 g。在经过亲和层析柱抽提后的8个留样组分中,组分2~5含有目的蛋白的粗提物。经过分子筛等方法除杂后,Bradford蛋白检测显示本次共提取蛋白总量345μg。SDS-聚丙烯酰胺凝胶电泳和银染方法结果证实此蛋白分子量大约为35~40 KDa。Westem Blot方法显示此蛋白与叶酸受体抗体特异结合。结论 使用本研究方法能够从人胎盘中成功提取并纯化人源的叶酸受体蛋白,每100 g胎盘可提取叶酸受体蛋白93μg。本研究为以后的叶酸受体抗体检测奠定了基础。更多还原

【Abstract】 Objective To establish the method of extraction,purification and identification of folate receptor from human Placenta.Methods A placenta was collected from a healthy woman when delivering in a hospital.The placental tissue was homogenized after cutting.Target protein was extracted by affinity chromatography from the homogenate after acidification and neutralization,then was purified by chemical and physical methods.The protein extraction was quantified by Bradford protein assay.The molecular weight was determined through sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver stain.Identification of the protein was performed by Western Blot.Results The wet weight of the placenta used for protein extraction was 370 g.Target protein was eluted into fraction2-5 among 8 fractions after extraction by affinity chromatography and was further purified.Results of Bradford protein assay showed that a total of 34 S μg protein was obtained.The molecular weight was about 35-40 KDa.Western Blot showed that the protein could specifically bind to the antibody to folate receptor.Conclusion The method of extraction,purification and identification of folate receptor from human placenta was successfully established.93 μg of folate receptor could be obtained from placenta per 100 g.The method in this study provides a good foundation for the detection of antibody to folate receptor.更多还原