人α_1微球蛋白基因的质粒载体构建和表达

Construction and Expression of the Plasmid Vector of the Human α_1-Microglobulin in Escharichia Coli

目的构建表达人α1-微球蛋白(α1-microglobulin,1α-MG)的重组质粒并纯化目的蛋白。方法从正常人肝脏中提取cDNA,RT-PCR扩增α1-mg基因,将扩增产物用NdeI/BamHI消化,切下目的基因片断克隆到质粒pET-15b中,构建重组pET-15b,转化入E.coliDH5α中,经酶谱分析和测序,鉴定出正确的重组质粒pET-15b。将重组pET-15b转化入宿主菌E.coliBL21(DE3)中,用IPTG进行诱导表达。提取细菌总蛋白质进行SDS-PAGE分析和蛋白印迹鉴定。利用重组蛋白中的6个组氨酸(His)组成的“标签”进行亲和层析,纯化重组蛋白。结果筛选到携带有α1-mg基因的正确重组质粒pET-15b,重组蛋白在BL21(DE3)pLysS中得到了诱导表达,含量达20mg/L细菌培养液。经一步金属螯合亲和层析纯化后,重组蛋白在SDS-PAGE上呈现出均一的单一条带。结论重组质粒pET-15b的构建和有活性的酶蛋白的表达成功说明,PCR扩增获得的α1-mg基因具有完整的阅读框架,亲和层析纯化后可得到活性和纯度均较高的目的蛋白,为研制国产免疫比浊法试剂盒打下基础。

Objective To construct a recombinant plasmid expressing theα1-microglobulin（α1-MG） from human liver and purify the target protein. Methods cDNA of human α1-mg was obtained by RT-PCR from the tissue of human liver. The gene was constructed imo the pET-15b expression vector and expression in Escharichia coli BL21 （DE3） after transformation and induction by IPTG. The complete protein of the host bacteria was extracted for SDS-PAGE. Afterwards the recombinant protein with an N-terminal His tag sequence was purified by affinity chromatography on a Ni^2＋ -Sepharose column. Results The expressed humanα1-MG in Escherichia coli was formed in inclusion body. After denaturation and renaturation, the inclusion protein became a soluble form with nature conformation. Conclusion The humanα1-MG was successfttUy expressed and purified, which can be used to get polyclone or monoclone antibody.