小鼠H_2B-GFP真核表达载体的构建与鉴定  

Construction and Identification of Mouse H_2B-GFP Eukaryotic Expressing Vector

作　　者：孙文靖[1] 马琳琳[1] 韩菲菲[1] 任聪[1] 傅松滨[1,2] 

机构地区：[1]哈尔滨医科大学医学遗传学研究室,150081 [2]黑龙江省生物医药工程重点实验室,哈尔滨150081

出　　处：《国际遗传学杂志》2006年第1期15-19,共5页International Journal of Genetics

基　　金：国家自然科学基金资助项目(No.30370783);黑龙江省卫生厅医学科研课题(No.2005-30);高等学校优秀青年教师教学科研奖励计划;教育部高等学校博士学科点专项科研基金(No.20040226001);哈尔滨医科大学研究生创新基金资助项目

摘　　要：目的构建小鼠H2B-绿色荧光蛋白(GFP)真核表达载体,为动态观察小鼠细胞中染色体的形态变化,从而进一步研究小鼠耐药细胞中双微体(DMs)的形成机制提供有效的分子工具。方法利用RT-PCR的方法获得小鼠H2B cDNA,以羧基端插入pcDNA3.1/CT-GFP-TOPO载体上,构建H2B-GFP真核表达载体,经菌液PCR、酶切及DNA测序鉴定插入片段大小、方向及序列的正确性,提取质粒转染小鼠胚胎成纤维细胞系NIH3T3进行鉴定。结果经菌液PCR、酶切和测序,证明小鼠H2B-GFP真核表达载体含有大小、方向及序列正确的H2B cDNA片段,转染NIH3T3后在细胞核中表达。结论成功构建了同时携带有G418筛选位点及多酶切位点的小鼠H2B-GFP真核表达载体,为其在小鼠体外培养细胞中的表达奠定了基础。Objective To construct a fluorescent eukaryotic expression vector coding mouse H2 B gene. Methods The mouse H2B cDNA was amplified by RT-PCR from the total RNA and inserted into pcDNA3.1/CTGFP-TOPO vector. The structure of the recombinant vector had been verified by PCR amplification, restriction analysis and sequencing. Then the reporting vector was transfected into mouse fibroblast cell line NIH3T3. Results The recombinant fluorescent expression vector coding mouse H2 B gene was correctly constructed in size, orientation and sequence, which was expressed in the nucleus of NIH3T3. Conclusion The recombinant mouse H2 B-GFP expression vector has been successfully constructed, which can help study the biochemical role of H2 B and chromosomes rnorphologic variation.

关 键 词：H2B 绿色荧光蛋白 真核表达载体 小鼠 

分 类 号：R346[医药卫生—基础医学]