吡喃糖氧化酶的原核表达及初步实验应用

Prokaryotic expression and initial experimental application of pyranose oxidase

目的原核表达、纯化、鉴定吡喃糖氧化酶(PROD),为其临床应用奠定基础。方法利用生物信息软件,比较不同种属的PROD核苷酸同源序列,优化设计PROD基因序列,委托上海捷瑞生物工程有限公司构建原核表达质粒pET15b-PROD,将此质粒导入大肠杆菌工程菌株BL21(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。其超声后的沉淀、上清液经镍离子柱亲和层析纯化、脱盐后进行活性测定,并配成应用液检测50例(30例糖尿病患者,20例健康对照)血液标本中的葡萄糖水平,分析其与葡萄糖氧化酶法(GOD法)和已糖激酶法(HK法)检测血糖诊断糖尿病的效能。结果重组质粒pET15b-PROD经测序PROD基因序列长度为1 869bp,表达623个氨基酸。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示该蛋白在相对分子质量68×103处有一条明显的条带,该条带在超声后沉淀中很弱,而在上清中较浓。上清液经纯化后的纯度大于90%,活性超过60kU/g,用其测定空腹血糖并用于糖尿病诊断的受试者工作特征曲线(ROC曲线)下面积(AUC)为0.995,用GOD法和HK法的AUC分别为0.994、0.992。根据约登指数确定最佳临界点,上述3种方法分别为7.11、7.16、7.06mmol/L。结论本研究生产的PROD以上清液中的表达为主,其纯度优、活性强、产量高,可用于血糖的检测。在本研究的基础之上可进一步优化条件使之应用于临床标本中1,5-脱水葡萄糖醇(1,5-AG)的检测。

Objective To express prokaryoticly,purify and identify pyranose oxidase(PROD)to lay the foundation for its clinical application.Methods The gene sequence of PROD was designed optimally by comparing nucleotide homologous sequences from various species by means of bioinformatics software.The prokaryotic expression plasmid of pET15 b-PROD was constructed by Shanghai Generay Biotech Co.,Ltd and expressed in E.coli engineering strain BL21(DE3)which was induced by isopropyl thio-beta-D-galactoside(IPTG).The precipitation and supernatant of this strain after ultrasonic processing was purified by Ni-NTA affinity chromatograph and desalinated,the activity of which was measured then,and the application liquid was prepared.The blood glucose concentrations of 50 patients(30 diabetic patients and 20 healthy controls)were detected by the application liquid,and the diagnostic efficiencies of glucose oxidase method(GOD method)and hexokinase method(HK method)were analyzed.Results The length of PROD gene sequence of recombinant plasmid of pET15 b-PROD was 1 869 bp,encoding 623 amino acids.SDS-PAGE profile showed an obvious protein band with a relative molecular weight of 68×103,and this band was very weak in precipitation,but fairly thick in supernatant by ultrasound.The purity and activity of the supernatant were more than 90% and 60 kU/g respectively.The area under curve(AUC)of the receiver operating characteristic(ROC)for the diagnosis of diabetes with fasting blood glucose concentration measured by using the purified supernatant was 0.995,while GOD and HK were 0.994,0.992,respectively.According to Youden index,the best cut-off points of fasting blood glucose were 7.11,7.16 and 7.06 mmol/L,respectively.Conclusion The PROD produced in this study is mainly expressed in the supernatant.It has high purity,high activity and high yield,and can be used for the detection of blood glucose.On the basis of this study,the conditions can be further optimized for the detection of 1,5-dehydrated glucosol(1,5-AG)in clinical specimens.