BPHL与PML-C间相互作用的胞内、外验证

目的 通过酵母双杂交实验及免疫共沉淀技术验证人联苯样水解酶(BPHL)与缺失核定位信号的人急性早幼粒细胞白血病(PML)基因的coiled-coil的结构域(PML-C)蛋白之间的相互作用。 方法 将表达PML-C诱饵蛋白及BPHL靶蛋白的重组质粒pGBKT7-PML-C及pACT2-BPHL共转化AH109酵母菌,通过酵母双杂交实验验证两者在活细胞内的相互作用。构建能在人胚肾293细胞中表达带HA标签的PML-C融合蛋白的重组载体pCMV-HA-PML-C,经酶切鉴定正确后,和表达带myc标签的BPHL融合蛋白的重组真核表达载体pCMV-myc-BPHL,共转染人胚肾293细胞,利用免疫共沉淀技术验证BPHL与PML-C间的相互作用。 结果 pGBKT7-PML-C及pACT2-BPHL质粒共转化AH109酵母菌后,可见蓝色阳性克隆。构建的重组表达载体pCMV-HA-PML-C及pCMV-myc-BPHL经双酶切鉴定正确后共转染HEK293细胞,抗HA多克隆抗体沉淀HA-PML-C相互作用蛋白复合物后,用抗c-myc单克隆抗体进行Western blot检测,可以检测到myc-BPHL的表达蛋白。 结论 成功构建了pCMV-HA-PML-C及pCMV-myc-BPHL融合蛋白真核表达重组载体,利用酵母双杂交实验及免疫共沉淀技术证实BPHL与PML-C之间存在着相互作用。

Identification of Interaction Between BPHL and PML-C

Objective To explore the interaction between BPHL and PML-C by co-immunoprecipitation and yeast two-hybird system. Methods The recombination expression plasmids pGBKT7-PML-C and pACT2-BPHL were cotransformed into yeast AH109, to investigate their interaction in vivo. The expression vector of HA-tagged fusion protein (pCMV-HA-PML-C) and the expression vector of myc-tagged fusion protein (pCMV-myc-BPHL) were constructed and identified respectively, and cotransfected into human embryo kidney 293 (HEK293) cells. the interaction between PML-C and BPHL was investigated by co-immunoprecipitation in vitro. Results Blue clones were found in QDO/5-bromo-4-chloro-3-indolyl-α-D-galactoside (X-α-gal) plate, eukaryotic expression vectors named as pCMV-HA-PML-C and pCMV-myc-BPHL were constructed and confirmed with double restriction enzyme digestion and co-transfected into HEK 293 cells successfully. After immunoprecipitation of HA-PML-C with anti-HA polyclonal antibody, expressed myc-BPHL protein was identified by Western blot with anti-c-myc monoclonal antibody from immunoprecipitated complex. Conclusion The eukaryotic expression vector of PCMV-HA-PML-C and PCMV-myc-BPHL were constructed successfully. The interaction between PML-C and BPHL was identified by co-immunoprecipitation and yeast two-hybird technique.