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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.
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