日本鳗鲡UNAG蛋白的克隆及其在非结合胆红素检测中的应用

UNAG cloning of Anguilla japonica and its application in the unconjugated bilirubin detection

血清中非结合胆红素含量升高与新生儿脑损伤的发展相关,但目前临床上没有方法可以直接测定非结合胆红素的血清水平.从日本鳗鲡(Anguilla japonica)中成功克隆了其UNAG基因,在原核细胞中成功表达UNAG重组蛋白,并通过Ni^(2+)-NTA进行纯化得到含量为1.8 mg/cm^3的UNAG目的蛋白,银染检测说明其纯度较高.利用该基因可以与非结合胆红素结合并发光的特异性,设计了一种新型的非结合胆红素检测方法,结果表明UNAG发光值与胆红素浓度呈良好的线性关系,回归方程为Y=17.96 X+4.333 3(R=0.994 9).该法可用于胆红素浓度的测定.

Increased serum levels of unconjugated bilirubin have been associated with the brain damage of newborns.However,there are no methods to directly determine the serum levels of unconjugated bilirubin in clinical.In this study,we cloned the UNAG from Anguilla japonica,which was then expressed in prokaryotic cell.The recombinant protein was purified by Ni^（2＋）-NTA,and the concentration reached 1.8 mg/cm^3 with high purity confirmed by silver staining method.Based on the characters that UNAG can be conjugated with unconjugated bilirubin and glow,a method for the detection of unconjugated bilirubin was established.Linear relationship was fine between the concentration of UC-BR and the fluorescence intensity of UNAG and the regression equation was established as Y =17.96X＋4.333 3（R=0.994 9）.The method can be applied for the detection of unconjugated bilirubin in clinic.