摘要
目的研究尿苷二磷酸葡萄糖醛酸基转移酶1A1的P364L突变型(c.1091 C>T)对催化非结合胆红素葡糖醛酸化的影响。方法构建含有UGT1A1编码序列的野生型和P364L突变型质粒,转染HEK293细胞,表达野生型与突变型蛋白;Western blot验证野生型和P364L突变型蛋白表达情况;超声裂解细胞提取表达蛋白;设计两组反应,野生型为对照组,P364L突变型为实验组,分别催化非结合胆红素反应,HPLC定量检测两组中非结合胆红素浓度变化情况。结果对照组中非结合胆红素的浓度随着时间的延长呈逐渐下降的趋势,而结合胆红素的浓度随时间延长逐渐升高;实验组中非结合胆红素浓度下降程度远小于对照组,未见明显的结合胆红素峰值;反应30 min时计算酶的活性P364L突变型为野生型的14.2%。结论突变酶UGT1A1-P364L催化非结合胆红素能力较野生型明显降低。
Objective To investigate the effect of UDP-glucuronosyhransferase 1A1 （ UGT1A1 ） genetic mutant P364L （c. 1091 C 〉 T） on the glucuronidation of unconjugated bilirubin. Methods The UGT1A1 wild-type and mutant P364L gene sequences were inserted into plasmid pEZ-M03 respectively, and then transfected into HEK293 cells. Western blotting was applied to test the expression of wild-type and mutant P364L-UGT1A1 proteins. Ultrasound was used to extract the intra-cellular compounds. High- performance liquid chromatography （HPLC） was used to detect the level of unconjugated bilirubin in both wild-type and mutant P364L catalytic reaction. Results The concentration of unconjugated bilirubin was significantly decreased while the peak concentration of conjugated bilirubin was increased over time in wild- type UGT1AI catalytic reaction. However, the concentration of unconjugated bilirubin was slightly decreased over time and the peak concentration of conjugated bilirubin was not obviously detected in P364L-UGT1A1 catalytic reaction. The mutant P364L-UGT1A1 contained only 14. 2% of wild-type enzyme activity. Conclusion Mutant P364L-UGT1A1 （1091C 〉 T） has significantly lower enzyme activity to catalyze unconjugated bi]irubin than wild-type UGT1A1.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2015年第10期1017-1021,共5页
Journal of Third Military Medical University
