A HUMAN APOLIPOPROTEIN AI-LIKE PROTEIN SECRETED BY YEAST

It has been suggested by many researchers that the dangers of developing atheroclerosis and the coronary heart disease in human race are closely correlated with the amount of high density lipoproteins in plasma. High density lipoprotein is capable of delivering cholesterol from the peripheral tissues to the liver for disposal and therefore prevent the accumulation of cholesterol in the artery wall. Apolipoprotein AI (apoAI) is the major polypeptide of high density lipoproteins and seems to play an important role in this cholesterol transport.

用酵母双杂交系统研究载脂蛋白AI(apoAI)和清道夫受体BI(SR-BI)间的相互作用

目的:利用酵母双杂交系统验证在胆固醇逆转运过程中起关键作用的大鼠载脂蛋白AI(apoAI)和清道夫受体BI- (SR -BI)间存在着相互作用,为初步筛选具有降脂活性组分提供1对新的靶点。方法:首先分别克隆了Wistar大鼠的apoAI和SR -BI基因的cDNA ,并构建了酵母表达载体,利用共转化技术观察到apoAI和SR -BI间存在着相互作用,并在酵母交配实验中证实了这个结果。结果:经共转化后的实验组与阳性对照组可在SD/ -Leu/- Trp/- His/- Ade/X -α- Gal平板上生长且菌斑呈蓝色,经测定α、β半乳糖苷酶活力可知酶活分别为8～1 2U和1 0～40U。酵母交配后的二倍体实验组、阳性对照组可在SD/- Leu/- Trp/ -His/- Ade/X -α- Gal平板上生长且菌斑呈蓝色。结论:apoAI和SR BI间的确存在相互作用。

大鼠SR-BI胞外域与ApoA-I蛋白相互作用的确认

先以含全长SR-BIcDNA序列的重组质粒pMD18-T-rS为摸板进行PCR反应扩增SR-BI得到胞外域cDNA片段,经测序证明正确后,定向克隆到酵母双杂交表达载体,然后与pGBKT7-ApoA-I质粒共转化酵母细胞,通过报告基因及酵母交配试验确认了SR-BI的胞外域部分和ApoA-I之间的确存,观察到ApoA-I与SR-BI胞外域间的相互作用力比与全长的SR-BI间的相互作用力提高了10%。