人M2型丙酮酸激酶促进肾癌细胞生长

Human Type M2 Pyruvate Kinase Promotes Renal Cancer Cell Growth

目的:构建带myc标签的人M2型丙酮酸激酶(PKM2)的真核表达载体,并瞬时转染肿瘤细胞,初步探讨PKM2的生物学功能。方法:以人乳腺文库为模板,采用PCR技术扩增PKM2基因,将其插入p XJ-40-myc载体,双酶切和基因测序验证后转染人胚肾293T细胞,Western印迹验证表达;将重组基因和空载体分别转染人肾透明细胞癌Caki-1细胞,CCK8法测定并绘制细胞生长曲线。结果:从人乳腺文库中获得长约1500 bp的DNA片段,连接到p XJ-40-myc载体上,测序结果与目的序列完全一致;转染人胚肾293T细胞后获得表达;细胞生长曲线结果显示,转染myc-PKM2的Caki-1细胞生长较空载体细胞快。结论:构建了myc-PKM2真核表达载体,为进一步研究PKM2在肿瘤发生发展中的作用奠定了基础。

Objective： To construct the eukaryotic expression vector of myc-tagged human type M2 pyruvate ki- nase（PKM2） and detect its activity in renal cancer ceils. Methods： Human PKM2 was amplified from human mammary cDNA library, and was inserted into eukaryotic expression vector of pXJ-40-myc. The recombinant plas- mid pXJ-40-myc-PKM2 was identified by enzyme digestion and sequnencing, transfected into the renal cancer cell line Caki-1 and detected by Western blot. Then CCK8 assay was performed to investigate the effect of myc- PKM2 on renal cancer cell proliferation. Results： The DNA fragment of about 1500 bp was successfully amplified by PCR, cloned into pXJ-40-myc, and identified by sequencing. Western blotting showed the expression of myc- PKM2 in the 293T cells. Cell growth curve demonstrated that ceils transfectd with myc-PKM2 grew significantly faster than those transfected with empty vector. Conclusion： The eukaryotic expression vector of myc-PKM2 was successfully constructed, which lays a molecular foundation for the study of PKM2 in renal cancer development and progression.