人α1微球蛋白基因克隆及其在大肠杆菌中的高效表达  

Cloning of human recombinant alpha l-microglobulin and expression in Escherichia coil

作　　者：魏雁虹[1] 耿辉[1] 宋帅[1] 孟莹[1] 杨广民[1] 

机构地区：[1]吉林省人民医院检验科,长春130021

出　　处：《中国医药》2013年第11期1597-1600,共4页China Medicine

基　　金：吉林省科技发展计划项目（20130102085JC）;吉林省卫生厅科研课题（20122078）

摘　　要：目的构建人α1微球蛋白（α1-MG）基因的原核表达质粒，在大肠杆菌中表达、纯化，为制备仅。一MG诊断试剂中原料抗体、标准品及质控品奠定基础。方法从人胚胎肝脏中提取总RNA，利用反转录聚合酶链反应技术扩增α1-MG基因。然后克隆至PQE30表达载体，转化大肠杆菌M15中进行表达，并经镍固定金属亲和层析进行纯化，蛋白质印迹进行免疫学鉴定。结果获得了与Genebank报道一致的仪。一MG基因片段，成功构建了成熟人α1-MG的原核表达载体PQE30-α1-MG，质粒转化大肠杆菌并经1mmol／L异丙基硫代半乳糖诱导5h后，可表达相对分子质量约25000的外源蛋白，表达的α1-MG大部分在包涵体中，并可经镍固定金属亲和层析纯化，纯化有效率95％以上，BCA法测定纯化的α1-MG蛋白含量为25mg／L。蛋白质印迹表明，该蛋白可与抗人仅．一MG天然抗体反应。结论通过基因工程方法可获得高效表达的重组α1-MG蛋白，该蛋白具备同天然仅。一MG相同的生物学特性，为下一步进行α1-MG相关研究及应用奠定基础。Objective To construct prokaryotic expression vector with gene of human alpha 1-microglobulin （ alphal -microglobulin, alphal-MG） and to obtain recombinanl human alphal-MG protein expressed in Escherichia coll. Methods Total RNA was extracted from human fetal liver and alphal-MG gene was amplified by reverse tran- scription-polymerase chain reaction（RT-PCR）. The gene was cloned into PQE30 expression vector and transformed into Escherichia coli M15. The expression of the target protein was induced with IPTG and purified by Ni^2 ＋ -NTA aga- rose column and identified by western blot method. Results We obtained the alphal-MG gene fragments consistent with those reported in Gene bank and successfully constructed prokaryotic expression vector of human alpha 1-MG. The protein was highly expressed in Escherichia coll. Most of the expression of alphal-MG was in inclusion bodies and could be purified by Ni^2＋ -NTA agarose column and the final protein purity reached above 95%. Western blot assay indicated that the monoclonal antibody against human native could react specifically with the recombinant pro- tein. Conclusion Recombinant α1 -MG protein has good biological characteristics with natural α1-MG which can be obtained by genetic engineering method.

关 键 词：Α1微球蛋白 重组蛋白 反转录聚合酶链式反应 蛋白质印迹 

分 类 号：Q78[生物学—分子生物学]