A standard curve for the product of SEAP is established, and the formula between the absorbance value and real value of SEAP is given. Then it is studied the formula from Michaelis constant of SEAP, reaction time, corresponding relationship between different amounts of mRNA and enzyme. In experiments, It is established the formula between the absorbance value and activity of SEAP at 405nm wavelength: y(enzyme activity) = (x(OD value) + 0. 002 8)/19.13 × 10; and the formula between the enzyme activity and absolute value of protein: 1U(enzyme activity)=5. 035 ng(value of protein), which is validated in the cell experiments and in vivo experiments of mice. It is proposed the method of improving the sensitivity of SEAP, and make the comparison among the efficiency of different methods in gene therapy experiments successfully. The standard can make SEAP reporter system raised from qualitative level to quantitative level, which sensitivity can meet the of 10-18mol.%通过建立SEAP(分泌性碱性磷酸酶)产物的标准曲线,得出SEAP的吸光值与酶活力、蛋白质绝对值之间相互换算的公式,从酶动力常数、酶反应时间、不同量mRNA与酶吸光值之间的对应关系等方面进行研究.建立了在405 nm波长处,SEAP的吸光值与酶活力之间、酶活力和蛋白质绝对值之间相互换算的两个公式:y(酶活力)=(x(吸光值)+0.002 8)/19.13×10;1U(酶活力)=5.035 ng(蛋白质绝对量),并在细胞和小鼠实验中得到了验证.建立了提高SEAP检测系统灵敏度的方法,并进行了导入目的基因不同方法的效率之间的比较.结果表明,建立的换算标准可以使SEAP报告基因系统从定性水平上升到定量水平,而且灵敏度可以达到10-18 mol的标准.
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