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目的构建带myc标签的自噬标志蛋白P62基因真核表达载体,获得myc-p62融合表达蛋白,并对其功能进行初步检测。方法利用聚合酶链式反应从乳腺文库中体外扩增人P62基因编码序列,将其与空p XJ-40-myc载体正确连接,重组质粒myc-p62转染HEK293T细胞,用Western blot法检测该融合蛋白的表达情况,并检测该蛋白对细胞外信号调节激酶(ERK)磷酸化的影响。结果构建得到myc-P62基因的真核表达载体,双酶切鉴定得到与预期片段大小相符的外源性基因插入片段,经测序与目的序列完全一致;经Western blot法检测,融合蛋白成功表达,并能够抑制ERK磷酸化。结论成功表达了myc-p62融合蛋白并能抑制ERK磷酸化。
Objective To construct eukaryotic expression vector carrying myc tagged P62 gene and obtain myc-p62 fusion protein, and to detect its function. Methods The coding sequence of human P62 gene was amplified from mammary gland by polymerase chain reaction and ligated with empty pXJ-40-myc vector. The recombinant plasmid myc-p62 was transfected into HEK293T cells. The expression of fusion protein and the effect of this protein on the phosphorylation of extracellular signal-regulated kinase (ERK) were examined. Results The eukaryotic expression vector of myc-P62 gene was constructed and double-digested to obtain the exogenous gene fragment corresponding to the expected fragment size. The sequence of the exogenous gene was exactly the same as that of the expected fragment. The fusion protein was successfully expressed by Western blot, And can inhibit ERK phosphorylation. Conclusion The myc-p62 fusion protein was successfully expressed and inhibited ERK phosphorylation.