可介导多药耐药的cfr基因的原核表达及多克隆抗体的制备

Prokaryotic expression of the multi-drug resistant cfr gene and preparation of polyclonal antibodies against the expressed protein

为建立监测Cfr蛋白的方法奠定基础,采用PCR方法扩增cfr基因,并克隆入pEasy-T载体;测序确认后克隆入pET-28a(+)载体,构建重组质粒pET-28a-cfr,然后转化入大肠杆菌BL21(DE3)中并用IPTG诱导表达Cfr蛋白;用纯化的Cfr蛋白免疫BALB/c小鼠制备多克隆抗体,并对抗体进行鉴定。结果显示,成功克隆了全长cfr基因,构建了重组质粒pET-28a-cfr,并诱导了Cfr蛋白表达以及制备了小鼠抗Cfr蛋白的多克隆抗体;Western-blot法鉴定该抗体可特异性识别重组蛋白Cfr;ELISA法测定抗体效价为1∶64 000。结果表明,用大肠杆菌BL21(DE3)能够成功表达Cfr蛋白,并且获得了高特异性、高效价的小鼠抗Cfr蛋白的多克隆抗体。

This study aims to provide a basis for establishment of immuno-method for diagnosis of Cfr protein.The whole coding sequence of cfr gene was amplified using PCR,then linked into pEasy-T vector and transformed into Escherichia coli Trans 5αcompetent cells.The cfr gene in recombinant pEasy-T vector was first confirmed by sequencing,and linked into the expression vector pET-28a(+).The cfr positive vector was transformed into E.coli BL21(DE3)competent cells to induce the expression of Cfr protein.BALB/c mice were immunized with purified Cfr protein to obtain the polyclonal antibodies against this protein.Western-blot analysis was used to detect the specificity of the polyclonal antibodies.ELISA was established to detect the sensitivity of polyclonal antibodies.The polyclonal antibodies of Cfr were obtained successfully.Western-blot analysis showed that the antibodies had good specificity.The titer of anti-Cfr polyclonal antibodies was 1∶64 000.In conclusion,Cfr protein was expressed in BL21(DE3)competent cells.The polyclonal antibodies against the Cfr protein were obtained.