Research And Development Of Technologies For The Purification And Detection Of Selenoprotein-P From Human Plasma

Selenoprotein-P is a plasma protein rich in Selenium, which takes up about fiftypercent of the whole Selenium in human plasma. So the Selenium concentration ofhuman plasma is heavily affected by Selenoprotein-P. The major functions ofSelenoprotein-P are transporting Selenium and having anti-oxidation activity. It’sreported that the low concentration of Selenoprotein-P in human plasma hasrelationship with KeShan disease, White Muscle Disease and some inflammations. Soit is important to investigate Selenoprotein-P further.In order to investigate Selenoprotein-P, Selenoprotein-P wih high purity must beabtained firstly. The concentration of Selenoprotein-P in plasma is very low, average3.04mg/L. So high specificity of the purification method is required. As reported theeffective method to abtain Selenoprotein-P is immunoaffinity chromatography. So,how to get the Selenoprotein-P samples which can generate monoclonal antibodybecomes the chief difficulty. But until now, it’s only reported that four steps ofchromatography were used to get Selenoprotein-P samples which can generatemonoclonal antibody, which employed complex processes and had a low recoveryabout four to eight percent. There is no effect method to get Selenoprotein-P samplesand no Selenoprotein-P monoclonal antibody can be bought. So, The use ofimmunoaffinity chromatography is restricted. The purpose of our research is todevelop an effective method to get enough Selenoprotein-P samples, which can beused to generate Selenoprotein-P monoclonal antibody.We developed a simple and effective method to get Selenoprotein-P samples.Two successive steps of affinity chromatography, i.e., Heparin-Sepharose andNi-Sepharose chromatography are developed to purify Selenoprotein-P from humanplasma. We have investigated the separation characters of domestic and GE packingmaterials. The GE packing materials with high activity chosen. The best steppedgradient elution method is established and the affinity chromatography method topurify Selenoprotein-P is formed. HG-AFS is used as the detection method ofSelenoprotein-P to monitor the separation procedues. We have accomplished themethod validation of the HG-AFS which is precise and has high sensitivity.SDS-PAGE is used to confirm that we have got Selenoprotein-P with a certain purity.At last, after ultrafiltration and lyophilization the Selenoprotein-P solid powder isabtained. An intact method to purify, detect and final treat the Selenoprotein-P iscompleted. Finally, solid Selenoprotein-P with a certain purity is abtained. Theproblem that it is difficult to get Selenoprotein-P samples is resolved. Our researchmade the foundation for the purification of Selenoprotein-P using immunoaffinitychromatography which also promote the further research of Selenoprotein-P.