摘要
目的原核表达果糖氨基酸氧化酶(FAOX),建立检测糖化血红蛋白A1c(HbA1c)的酶法并作初步评价。方法构建重组质粒pET32a(+)/FAOX,转化大肠埃希菌Rosetta(DE3),获得重组FAOX;以糖基化六肽为底物检测酶活性;初步建立检测HbA1c的酶法并进行方法学评价。结果重组FAOX在Rosetta(DE3)中高效表达,SDS-PAGE电泳分析显示其相对分子质量(Mr)约65 000;酶比活性为22 U/mg;建立的检测HbA1c酶法的批内、批间、日间CV均<5%;低值(5.1%)和高值(15.8%)HbA1c的回收率分别为98.1%和98.9%;总胆红素(<220μmol/L)、三酰甘油(<10.8 mmol/L)和维生素C(<500 mg/L)对酶法检测HbA1c无影响;与HPLC法和HA-8160型全自动糖化血红蛋白仪测定结果进行比较,相关系数分别为0.977和0.993。结论 FAOX可在大肠埃希菌Rosetta(DE3)中高效表达,用其建立的检测HbA1c的酶法可满足临床对HbA1c测定的要求。
Objective To express fructosyl amino acid oxidase （FAOX） in prokaryocytes for establishing an enzymatic method to de- termine glycosylated hemoglobin A1 c （HbA1 c） and evaluate its methodology. Methods The recombinant plasmid pET32a （ ＋ ）/ FAOX was constructed and transformed into E. coli Rosetta （DE3） to obtain recombinant FAOX after expression and purification. The glycated hexapeptides was used as substrate to determine the activity of recombinant FAOX. The enzymatic method for determination of HbAlc was established and its methodology was evaluated. Results The recombinant FAOX was highly expressed in E. coli Rosetta （DE3）. The relative molecular weight of FAOX was about 65 000 as shown on SDS-PAGE and its specific activity was 22 U/mg. The coefficients of variation （CV） for within-run, between-run and inter-day assay of the enzymatic method in determining HbAlc were all less than 5%. The recovery rates of HbAlc at low value （5.1%） and high value （ 15.8% ） were 98.1% and 98.9% respectively. No interference of total bilirubin （ 〈 220 μmol/L）, triglyceride （ 〈 10.8 mmol/L） and vitamin C （ 〈 500 mg/L） on the determination was observed. The results of the enzymatic method showed close correlation with those of HPLC assay and HA-8160 automated glycated hemoglobin analyzer. Conclusion FAOX could be highly expressed in E. coli Rosetta （ DE3 ）. The enzymatic method developed in this study should be satisfied for clinical requirements in HbA1 c determination.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2013年第2期83-86,共4页
Chinese Journal of Clinical Laboratory Science
关键词
果糖氨基酸氧化酶
原核表达
糖化血红蛋白
fructosyl amino acid oxidase
prokaryotic expression
glycosylated hemoglobin
