Isolation And Purification Of 33.5kDa Versicular Protein From Human Bile

33.5kDa vesicular protein in bile was separated and purified from cholesterol gallstones patients by Paokin Ma in 1998. Its protein concentration was 0.46±0.06mg/ml and occupied by 0.552% in total protein in bile. Though the amount of 33.5kDa vesicular protein is even small, it has a remarkable function to help forming cholesterol nuclear. Previous research suggested that the activity of forming nuclear strongly reached to 0.310. It is the most active pro-nucleating protein ever discovered. To find the best pro-nucleating protein, find out its properties and develop detection kit, judge and predict the develop trend of gall-stone are the final purposes of gall-stone cause researching.This experiment adopted several methods such as centrifuging,ion-exchange chromatography,molecular sieve chromatography to find a fast,easy and economic way to separate and purify 33.5kDa vesicular protein from bile. In base of that, we can use purified 33.5kDa vesicular protein to prepare monoclonal antibody. Thus we can make golden colloidal fast detection kit to detect protein as general diagnostic standard.First of all, anionic ion-exchange chromatography and cationic ion-exchange chromatography were applied in our work, the desire protein was acidity after fumbled four pH. So the protein have adsorption effect to filler but can't find the condition of elute.Then,we adopted Phenyl-Sepharose Fast Flow, the result was not good for the bilirubin's hydrophobicity was better than 33.5kDa vesicular protein. At last, we got the result through centrifuging combine molecular sieve chromatography. We determined the protein active through observing the promoting effect in model bile and got a result that the protein sample have some pro-nucleating effect. After this experiment we determined the protein purification with RP-HPLC. The next step, we will do some experiments to analyse the sample.