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  

Construction of Human VHL Eukaryotic Expression Vector and its Suppressive Role in Cancer Cell Growth

作　　者：李玲[1] 王婷 梁迎春[1] 张立 冯滢滢[1] 周丽英[1] 冀全博[1] 郭靖[1] 徐小洁[1] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]陕西省妇幼保健院辅助生殖中心,陕西西安710003 [3]总参警卫局卫生保健处,北京100017

出　　处：《生物技术通讯》2014年第6期775-778,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31100604);北京市科技新星计划(Z141102001814055);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：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p XJ-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Hep G2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p XJ-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。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.

关 键 词：人VHL基因 克隆 真核表达 肿瘤细胞 

分 类 号：Q78[生物学—分子生物学]