论文部分内容阅读
目的使用免疫共沉淀技术法检测人p65和SMRT蛋白在儿童急性髓细胞白血病(AML)细胞株THP-1中相互作用。方法体外人工合成p65基因CDS区碱基序列和SMRT-C645bp对应的基因片段,分别克隆至pUC57载体,经酶切、测序正确后,PCR扩增目的基因片段,酶切后亚克隆至带有HA标签和Myc标签的真核表达载体pCMV-HA和pCMV-Myc,测序、酶切鉴定正确后,共转染THP-1细胞,免疫共沉淀法检测二者间相互作用。结果成功构建了人p65和SMRT蛋白真核表达载体pCMV-HA-p65和pCMV-Myc-SMRT,共转染THP-1细胞,抗HA和Myc多克隆抗体分别沉淀细胞裂解液,用抗Myc和HA单克隆抗体分别进行Western Blotting检测,可以检测到HA-p65和Myc-SMRT蛋白的表达。结论成功构建了人p65和SMRT蛋白重组真核表达载体,在THP-1细胞株中表达后,免疫共沉淀证实p65和SMRT蛋白间存在相互作用,为进一步探讨儿童AML发病机制和白血病的靶向治疗提供理论依据。
Objective To detect the interaction of human p65 and SMRT proteins in childhood acute myeloid leukemia (AML) cell line THP-1 by co-immunoprecipitation. Methods The gene sequence of CDS region of p65 gene and SMRT-C645bp gene were synthesized in vitro and cloned into pUC57 vector respectively. After digestion and sequencing, the target gene fragment was amplified by PCR. After digestion, The eukaryotic expression vectors pCMV-HA and pCMV-Myc with the tag and Myc were sequenced and identified by restriction enzyme digestion. The THP-1 cells were co-transfected and the co-immunoprecipitation assay was used to detect the interaction between them. Results The eukaryotic expression vectors pCMV-HA-p65 and pCMV-Myc-SMRT of p65 and SMRT were successfully constructed and transfected into THP-1 cells. Anti-HA and Myc polyclonal antibody were respectively used to precipitate cell lysate. HA monoclonal antibody were detected by Western Blotting, HA-p65 and Myc-SMRT protein expression can be detected. Conclusion The recombinant eukaryotic expression vector of human p65 and SMRT protein was successfully constructed. After THP-1 cell line was expressed, co-immunoprecipitation confirmed the interaction between p65 and SMRT protein. To further explore the pathogenesis of childhood AML and the target of leukemia Provide a theoretical basis for treatment.