c-Jun氨基末端激酶3结构域突变质粒的构建、鉴定与转染

为构建c-Jun氨基末端激酶3 (c-Jun N-terminal kinase3,JNK3)的p VP16-eGFP-Myc-JNK3活化环(activation-loop,A-loop)结构域突变型重组质粒,通过A-loop突变型JNK3的c DNA序列以及p VP16-eGFP-Myc的酶切位点设计引物,PCR扩增目的基因,使用限制性内切酶Mlu I与Xba I将基因克隆到p VP16-eGFP-Myc真核表达载体中,构建p VP16-eGFP-Myc-JNK3(A-loop)结构域突变型重组质粒。转化后,通过双酶切鉴定与DNA测序判断是否成功构建重组质粒,使用Trans IntroTMEL Transfection Reagent转染人胚肾(human emborynic kidney,HEK) 293T细胞,通过荧光显微镜下观察融合蛋白表达情况。结果表明:构建p VP16-eGFP-Myc-JNK3 (A-loop)结构域突变型重组质粒,双酶切鉴定和测序结果显示构建成功; p VP16-eGFP-MycJNK3 (A-loop)成功转染HEK293T细胞且表达。鼠源JNK3结构域突变型质粒构建成功,对进一步研究JNK3结构域在相关疾病的作用和机制提供实验工具。

Construction, Identification and Transfection of JNK3 Domain Mutant Plasmid

In order to construct the domain mutant recombinant plasmid pVP16-eGFP-Myc-JNK3 Activation-loop (A-loop) of c-Jun N-terminal kinase 3 (JNK3). The DNA sequence of A-loop mutant JNK3 and the restriction site of pVP16-eGFP-Myc were used to design primers, the target gene was amplified by PCR and cloned into eukaryotic expression vector by restriction endonuclease Mlu I and Xba I, the mutant recombinant plasmid of pVP16-eGFP-Myc-JNK3 (A-loop) domain was constructed. After conversion, the recombinant plasmid was identified by double-enzymeSdigestion and DNA sequencing, human embryonic kidney(HEK) 293T cells were transfected with TransIntro^TM EL Transfection Reagent and the expression of fusion protein was observed under fluorescence microscope. The results show that the mutant recombinant plasmid pVP16-eGFP-Myc-JNK3 (A-loop) was constructed, the results of double-enzymeSdigestion and sequencing analysis showed that the construction was successful, pVP16-eGFP-Myc-JNK3(A-loop)is successfully transfected into cells and expressed. Successful construction of mutant plasmid of mouse-derived JNK3 domain provides an experimental tool for further studying the role and mechanism of JNK3 domain in related diseases.