白介素1受体相关激酶4重组过表达质粒pcDNA3.1-IRAK4的构建及转染

Construction of Recombinant IRAK4 Overexpressed Plasmids and Validation of Their Transfection to H9c2 Cardiomyoblasts

目的构建重组过表达质粒pc DNA3.1-IRAK4并检验其在H9c2细胞中的表达。方法在全球科学家质粒共享非盈利组织Addgene申请到p DONR223-IRAK4质粒菌种,摇菌提取质粒后测序。设计引物将目的片段扩增并跑胶回收。将目的片段PCR扩增产物和空质粒pc DNA3.1采用Bam HⅠ和EcoRⅠ酶切,构建重组表达质粒。将重组质粒使用两种转染试剂转染入H9c2细胞中,并借助Western blot法检测IRAK4蛋白在H9c2细胞中的表达。结果重组过表达质粒经菌落PCR、序列测定证实,扩增基因片段IRAK4与基因数据库中序列一致,并且含有终止密码子。转染入H9c2细胞中能够过表达,表明IRAK4重组过表达质粒构建成功。结论成功构建IRAK4基因过表达重组质粒,获得外源性IRAK4转染的H9c2细胞。

Objective To construct the recombinant IRAK4 overexpressed plasmids and transfect them in H9c2 cardiomyoblasts. Methods pDONR223 - IRAK4 plasmids in Bacteria were provided for free by Addgene. Vector NTI was used to design the special primer of IRAK4, which expanded the genetic segment of IRAK4 by RT - PCR. The augment segment of IRAK4 and plasmid pcDNA3.1 were digested with endonuclease BamH I and EcoR I , then to connect. The recombinant overexpressed plasmid was transfected into H9c2 cardiomyoblasts, then to assay the expression of IRAK4 protein by Western blot. Results The augment segment was the IRAK4 genetic segment with stop eodon identified by RT- PCR, electrophoresis and sequence identification, which was in accordance with the sequence published in the gene bank, and the recombinant plasmid can overexpress in the H9c2 cardiomyoblasts, showing that the recombinant plasmid of IRAK4 was constructed successfully. Conclusion The recombinant plasmid of IRAK4 was successfully constructed and transleered into H9c2 cardiomyoblasts.