新耐药基因HA117多克隆抗体的制备与鉴定

Preparation and identification of polyclonal antibodies of novel multidrug resistance gene HA117

目的制备HA117多克隆抗体。方法以HA117表达质粒pAdTrack-HA117为模板,PCR扩增HA117基因,扩增产物转入BL21(DE3)感受态细胞,进行HA117蛋白诱导表达,采用SDS-PAGE检测表达产物。纯化HA117蛋白后免疫新西兰大白兔,制备多克隆抗体。通过间接ELISA检测其效价。将转染携带HA117重组腺病毒的HEK293细胞为实验组,未转染的HEK293细胞为对照组,用细胞免疫荧光和Western blot方法检测其过表达的HA117蛋白,验证制备的HA117多克隆抗体的特异性。结果成功获得HA117多克隆抗体,间接ELISA法检测显示抗HA117血清效价为1∶729 000。细胞免疫荧光和Western blot检测示制备的HA117多克隆抗体可以特异性识别HA117抗原蛋白。结论获得了效价和特异性较高的HA117多克隆抗体。

Objective To prepare polyclonal antibodies of a novel multidrug resistance gene,HA117.Methods pAdTrack-HA117 plasmid was used as the template to amplify the total cDNA of HA117 by using PCR.The products of PCR were transferred into BL21（DE3） competent cells to induce the protein expression of HA117 and identified by SDS-PAGE.New Zealand rabbits were immunized with HA117 protein after been purified in order to get the polyclonal antibodies of HA117.Indirect ELISA was used to detect the titer of the polyclonal antibodies of HA117.Adenovirus vector encoding HA117 gene was transducted into HEK293.Immunofluorescence staining and Western blot analysis were used to detect the specificity of polyclonal antibodies.Results Polyclonal antibodies of HA117 were obtained successfully.The anti-HA117 serum titer was 1∶729 000 by indirect ELISA.The polyclone antibody of HA117 identified the HA117 protein in transducted HEK293 specifically by immunofluorescence staining and Western bolt analysis.Conclusion Our prepared polyclonal antibodies of HA117 are of high titer and high specificity.