对氧磷脂酶PON1蛋白的原核重组表达  

Construction and expression of PON1 fusion protein

作　　者：郝雪微[1] 李淑珍[1] 吕鑫钰[1] 朱大岭[1] 

机构地区：[1]哈尔滨医科大学药学院生物制药学教研室,黑龙江哈尔滨150081

出　　处：《哈尔滨医科大学学报》2012年第3期203-206,共4页Journal of Harbin Medical University

基　　金：黑龙江省教育厅科学研究技术项目(11551275)

摘　　要：目的重组及表达对氧磷脂酶PON1的融合蛋白,为进一步制备相应单克隆抗体作准备,为建立一种冠心病辅助诊断方法奠定基础。方法以成人肝cDNA文库为模板,应用PCR技术扩增出PON1基因,将其分别与带有HIS标签的pET-32a及GST标签的pGEX-4T-1载体连接,转化入DH5α菌中进行克隆并测序鉴定。进一步构建表达体系,用异丙基β-D-硫代半乳糖苷(IPTG)诱导,最终在BL21菌中表达PON1融合蛋白(分别含有HIS、GST-Tag),并进行SDS-PAGE及Western blot鉴定。结果以成人肝cDNA文库为模板经PCR扩增后得到一条234 bp的DNA片段,经测序鉴定为PON1基因序列;并在大肠杆菌中实现了包涵体形式的表达,经Western blot鉴定为PON1融合蛋白。结论采用原核表达方法可获得高纯度的PON1融合蛋白,为进一步制备相应的单克隆抗体和开发PON1诊断试剂盒打下基础,为冠心病的诊断开辟新途径。Objective To recombine and express of human paraoxonase-1 （ PON1 ）fusion pro- tein, then purify for further preparation of its monoclonal antibody in order to establish an early diagnostic method of coronary heart disease（CHD）. Methods The gene encoding PON1 was cloned by using PCR techniques from eDNA of adult liver, and then the amplified gene was inserted into vectors pET-32a and pGEX-4T-1, then subcloned into DH-5a. The recombinant plasmids were constructed and transformed into E. coli BL21 cells,then protein expression （including His-Tag and GST-Tag, respectively） were induced by IPTG and identified by SDSPAGE and Western blot. Results A band of DNA about 234 bp by DNA sequencing was con- firmed as PON1 gene, the expression system was constructed, and soluble expression of PON1 fusion protein was achieved successfully, which was identified by Western blot. Conclusion Fusion protein PON1 with high purity could be expressed in prokaryotic system. The study does a further preparation of its monoclonal antibody and lays a foundation for opening up new ways for early diagnosis of coronary artery diseases.

关 键 词：冠心病 生物标志物 原核表达 

分 类 号：R541.4[医药卫生—心血管疾病] Q78[医药卫生—内科学]