人VHL基因真核表达载体的构建及其对肿瘤细胞生长的抑制

摘要

Objective: To construct the eukaryotic expression vector of myc-tagged human von Hippel-Lindau (VHL) gene and examine activity of its product in cancer cells. Methods: Human VHL gene was amplified from human mammary cDNA library, and was inserted into eukaryotic expression vector of pXJ-40-myc, followed by en⁃zyme digestion and sequencing confirmation. The resulting plasmid was transfected into HEK293T cells, and its ex⁃pression was detected by Western blotting. Effect of myc-VHL on cancer cell growth was investigated by CCK8 as⁃say, using breast cancer and liver cancer cells. Results: The DNA fragment about 650 bp was amplified by PCR, and verified by sequencing after it was cloned into pXJ-40-myc. Western blotting showed the expression product of myc-VHL with Mr of 26 kD in the HEK293T cells. Cell growth curve demonstrated that cells transfected with myc-VHL grew significantly slower than those transfected with empty vector. Conclusion: The eukaryotic expres⁃sion vector of myc-VHL was constructed, and its product played suppressive role on cancer cell growth, based on which, VHL in cancer development and progression will be studied.%目的：构建人抑癌基因VHL的真核表达载体，并验证其对肿瘤细胞生长的影响。方法：采用PCR技术从人乳腺文库中扩增人VHL基因，将其克隆到pXJ-40-myc载体中，酶切和测序验证后转染人胚肾293T细胞，通过蛋白免疫印迹鉴定其表达；转染人乳腺癌ZR75-1细胞和肝癌HepG2细胞，通过CCK8法测定细胞生长曲线。结果：从人乳腺文库中扩增得到约650 bp的DNA片段，并克隆至pXJ-40-myc载体上，且测序与目的序列完全一致；转染人胚肾293T细胞后，蛋白免疫印迹检测到相对分子质量为26×103的目的基因表达产物；细胞生长曲线显示，转染myc-VHL的乳腺癌、肝癌细胞较空载体细胞生长慢。结论：构建了myc-VHL真核表达载体，myc-VHL抑制癌细胞生长，为进一步研究VHL在肿瘤发生发展中的功能奠定了基础。