人胱抑素C的原核表达及活性鉴定  

Prokaryotic expression and activity identification of human cystatin C

作　　者：李原[1] 刘如石[2] 蒋琰[1] 李桑[3] 郑姣[3] 何赛[3] 吴意[1] 

机构地区：[1]湖南师范大学第一附属医院检验科,长沙410005 [2]湖南师范大学医学院医学分子与免疫诊断实验室,长沙410013 [3]湖南师范大学生命科学院,长沙410081

出　　处：《检验医学与临床》2016年第6期730-733,共4页Laboratory Medicine and Clinic

基　　金：湖南省科技计划项目(2014SK3100)

摘　　要：目的建立胱抑素C蛋白(Cys C)原核表达系统,表达、纯化并鉴定重组的人Cys C。方法在不改变氨基酸序列的前提下,对Cys C的编码基因进行大肠杆菌同义密码子偏好性优化后,人工合成其序列,PCR扩增Cys C基因,在原核系统中表达重组人Cys C。纯化表达的Cys C重组蛋白,采用间接ELISA法测定其反应灵敏度及免疫原性。结果十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定可知,重组基因在大肠杆菌中获得了有效表达,相对分子质量约为35×103,Western blot结果进一步说明重组蛋白为Cys C蛋白,间接ELISA结果显示,在包被浓度为0.5mg/L时便产生了阳性值。结论成功建立了Cys C原核表达系统,Cys C蛋白作为抗原具有良好的免疫反应性与免疫原性,为制备相应的抗原诊断单克隆抗体打下了基础,同时也为开发Cys C快速诊断ELISA试剂盒提供了可选原料。Objective To establish a prokaryotic expression system of cystatin C（Cys C）protein for expressing,purifying and identifying recombinant human Cys C protein.Methods Under the premise of unchanging amino acid sequence,the Cys C coding gene was performed the Escherichia（E.）coli synonym codon bias optimization and its sequence was artificially synthesized.The Cys C gene was amplified by PCR.The human recombinant Cys C protein was expressed in the prokaryotic system.The expressed Cys C recombinant protein was purified.Its reaction sensitivity and immunogenicity were assayed by indirect ELISA.Results The SDS-PAGE identification indicated the recombinant gene gained the effective expression in E.coli,the molecular weight of recombinant Cys C protein was 35×103.The Western blot results further revealed that the recombinant protein was Cys C protein,the indirect ELISA results displayed that when the coating concentration was 0.5mg/L,the positive value would be produced.Conclusion The prokaryotic expression system is successfully established,Cys C protein as antigen has better immunoreactivity and immunogenicity,which lays a foundation for preparing corresponding antigen diagnostic monoclonal antibody,meanwhile also provides the optional raw material for developing Cys C rapid diagnosis ELISA reagent kit.

关 键 词：人胱抑素C 酶联免疫吸附试验 免疫反应性 免疫原性 

分 类 号：R573.1[医药卫生—消化系统]