重组人烯醇酶-α的原核表达及生物信息学分析  

Prokaryotic expression and biological characteristics of recombinant human enolase-α

作　　者：王波[1] 刘思雨 罗红[2] WANG Bo;LIU Si-yu;LUO Hong(Department of Clinical Medicine Laboratory,Dalian Municipal Central Hospital,Dalian 116033;College of Medical Laboratory,Dalian Medical University,Dalian 116044,China)

机构地区：[1]大连市中心医院检验科,辽宁大连116033 [2]大连医科大学检验医学院,辽宁大连116044

出　　处：《基础医学与临床》2019年第9期1300-1304,共5页Basic and Clinical Medicine

基　　金：大连市医药卫生科学计划(1511015)

摘　　要：目的通过原核表达技术获得人烯醇酶-α(ENO1)重组蛋白,并对表达产物进行生物信息学分析及抗体结合测定,为应用血清学检测技术进行肝纤维化早期诊断奠定基础。方法根据GenBank中登录的ENO1序列(cDNA clone BC015641,MGC:23319 IMAGE:4643088),设计特异性引物,以ENO1的cDNA为模板,用PCR技术扩增ENO1序列,构建该基因的克隆载体pEASY-T1/ENO1及表达载体pET-32a(+)/ENO1,通过IPTG诱导表达重组蛋白rENO1。利用生物信息学相关软件(ProtParam、ProtScale、SignalP 4.1 server、Signal-3L、TMpred、DAS、NetPhos 2.0 Server)分析人ENO1蛋白的理化性质、信号肽、跨膜域及磷酸化位点等信息,并通过Western blot检测其与相应抗体的反应性。结果成功构建重组质粒pEASY-T1/ENO1及表达载体pET-32a(+)/ENO1,构建的克隆质粒测序结果与GenBank中登录的ENO1序列的比对符合率为100%。获得的ENO1融合蛋白分子质量约为67 ku。该ENO1片段由434个氨基酸组成,其相对分子质量为47 168.96,理论pI为7.01,是一种稳定的亲水性蛋白。该蛋白与相应抗体具有良好的反应性。结论重组人烯醇酶-α可以通过原核表达技术获得。获得的烯醇酶-α融合蛋白片段为稳定的亲水性胞外蛋白,可以与相应抗体反应,为其作为相关标志物用于肝纤维化的早期快速诊断奠定基础。Objective To collect human enolase-α(ENO1)recombinant protein by prokaryotic expression technology,and carry out bioinformatics analysis of the expressed product.Methods According to the ENO1 gene sequence(cDNA clone BC015641 MGC:23319 IMAGE:4643088)contained in GenBank,the specific primers of the gene fragment were designed.The ENO1 sequence was amplified by PCR and the clone of the gene was constructed by using the cDNA of ENO1 as a template.The vector pEASY-T1/ENO1 and the expression vector pET-32a(+)/ENO1 were induced to express the recombinant protein ENO1 by IPTG.The bioinformatics related software(ProtParam,ProtScale,SignalP 4.1 server,Signal-3L,TMpred,DAS,NetPhos 2.0 Server)was used to analyze the physicochemical properties,signal peptides,transmembrane domains and phosphorylation sites of the recombinant protein ENO1.Results The recombinant plasmid pEASY-T1/ENO1 and the expression vector pET-32a(+)/ENO1 were successfully constructed.The coincidence rate of the cloned plasmids and the ENO1 gene sequence in GenBank was 100%.The obtained rENO1 was about 67 ku.The ENO1 fragment consisted of 434 amino acids with a relative molecular mass of 47 168.96 and a theoretical pI of 7.01,which is a stable hydrophilic protein.Conclusions Human enolase-αcan be obtained by prokaryotic expression technology.The obtained fusion protein fragment is a stable hydrophilic extracellular protein,which lays a foundation for rapid early diagnosis of liver disease related markers.

关 键 词：烯醇酶-α 原核表达 生物信息学 

分 类 号：R34[医药卫生—基础医学]