人H-ras基因真核表达载体的构建及其对肿瘤细胞生长的促进作用  

Construction of Human H-ras Eukaryotic Expression Vector and its Proliferative Role in Cancer Cell Growth

作　　者：张云静[1,2] 冯滢滢[2] 徐小洁[1] 王涛[1] 张柯[3] 梁迎春[1] 张蓉[2] 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]第二炮兵总医院,北京100088 [3]军事医学科学院科技部,北京100850

出　　处：《生物技术通讯》2015年第2期190-193,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81472589;31100604);北京市自然科学基金(7132155);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:构建癌基因H-ras真核表达载体,并检测其对肿瘤细胞生长的作用。方法:以乳腺文库为模板,PCR扩增H-ras基因片段,将其插入p XJ-40-myc载体后转染人胚肾HEK293T细胞,用Western印迹检测该融合蛋白的表达;将重组质粒转染人肠癌HCT-116细胞和人肝癌Hep G2细胞,通过细胞生长曲线(CCK8法)对myc-H-ras影响肿瘤细胞生长的情况进行分析。结果:利用PCR技术,从乳腺文库中扩增出H-ras基因片段,并成功插入p XJ-40-myc载体;重组质粒转染HEK293T细胞,经Western印迹检测融合蛋白正确表达;细胞生长曲线结果显示,转染myc-H-ras的结肠癌HCT-116细胞和人肝癌Hep G2细胞比转染空载体的细胞生长快。结论:构建了人H-ras基因真核表达载体,为进一步研究ras基因在肿瘤细胞中的作用奠定了一定基础。Objective： To construct the eukaryotic expression vector of myc-tagged human H-ras and detect its function in cancer cells. Methods： Human H-ras gene was amplified from the mammary library by the technique of PCR, and was inserted into eukaryotic expression vector of pXJ-40-myc. The recombinant plasmid pXJ-40-myc-H-ras was identified by sequencing, and transfected into HEK293T cells. The myc-H-ras protein was detected by Western blot. Then CCK8 assay was performed to investigate the effect of myc-H-ras on proliferation of colon cancer cell HCT-116 and liver cancer cell HepG2. Results： The DNA fragment of about 600 bp was successfully amplified by PCR, cloned into pXJ-40-myc, and identified by sequencing. Western blot showed the expression of myc-H-ras in the HEK293T cells. Cell growth curve demonstrated that cells transfected with myc-H-ras grew significantly faster than those transfected with empty vector. Conclusion： The eukaryotic expression vector of myc-H-ras was successfully constructed, which laid a foundation for the study of H-ras in cancer development and progression.

关 键 词：H-RAS基因 真核表达载体 肿瘤细胞 

分 类 号：Q78[生物学—分子生物学]