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电话:(手机号) 基金项目:四川省青年科技基金项目(08ZQ026-081) 作者简介:李雪婷(1974-),女,四川南充人,硕士研究生,讲师,主要从事食管癌基研 究.

E-mail:[email protected] 通讯作者:谢勇恩(1967-),男,四川南充人,博士,教授,主要从事食管癌研究. E-mail:[email protected] 收稿日期:2012-02-08(编辑部填写) 说明:作者对本刊投稿,请一律参照本刊《投稿须知》、或近期已发表文章格式、或以下范文格式修改后发送电子档.本刊其他特殊要求: 1.参考文献一般需要12条及以上,尽量引用本刊文献,并尽量引用近两年本文文献1-2条;

2.热烈欢迎各类基金课题稿件,对厅局级及以上课题论文优先发表. MBP-1分子真核表达载体的构建及其在食管癌细胞株Eca109中的高表达 李雪婷,谢勇恩 (川北医学院病理生理学教研室,四川 南充 637007) 【摘要】 目的:初步探讨MBP-1的分子功能.方法:通过PCR扩增获得MBP-1 基因编码序列,将其定向插入质粒pcDNA3.1(+)构建重组质粒pcDNA3.1-mbp-1,重组质粒转染体外培养的食管癌Eca109细胞株,Western-blotting检测MBP-1在食管癌细胞中的表达.结果:筛选出分子量较大的重组质粒,酶切分析和DNA测序分析证实重组质粒含MBP-1 基因完整编码序列,重组质粒转染食管癌Eca109细胞株后呈现高表达,其表达量约为对照细胞的2.1倍.结论:成功构建了MBP-1分子真核表达载体并在食管癌细胞中实现了高表达,为进一步深入研究MBP-1的分子功能奠定了基础. 【关键词】MBP-1;

真核表达载体;

食管癌细胞 Construction of the eukaryotic expressing vector for MBP-1 and its expression in esophageal cancer cells LI Xue-ting, XIE Yong-en (Department of Pathophysiology, North Sichuan Medical College, Nanchong 637007, Sichuan, China) 【Abstract】Objective: To preliminarily study the molecular function of MBP-1 molecule. Methods: MBP-1 gene fragment was obtained by polymerase chain reaction (PCR). MBP-1 gene was inserted into plasmid pcDNA3.1(+) to construct the recombinant eukaryotic expressing vector for MBP-1. Esophageal cancer cells were transfected with recombinant vector. MBP-1 expression was detected by using Western-blotting. Results: A recombinant plasmid was obtained during screening. The recombinant plasmid contains the coding sequence of MBP-1 gene, which was identified by restriction enzyme analysis and DNA sequencing. Western-blotting analysis showed that MBP-1 overexpressed in esophageal cancer cells with recombinant plasmid transfection. Conclusion: A recombinant eukaryotic expressing vector for MBP-1 was successfully constructed. MBP-1 overexpressed in esophageal cancer cells after transfection with the recombinant plasmid. These results provided the basis for further study of the molecular function of MBP-1. [Key words] MBP-1;

Eeukaryotic expressing vector;

Esophageal cancer cells MBP-1,又称为c-Myc启动子结合蛋白(c-Myc promoter binding protein),我们通过实验研究发现人食管上皮细胞在高浓度亚硝酸盐冲击诱导培养后MBP-1呈现下调表达[1],由于MBP-1可以调控包括c-Myc在内的多个肿瘤相关基因的表达,是一个值得深入研究的调控分子.本研究构建了MBP-1分子真核表达载体并在食管癌细胞株Eca109中实现了高表达,为进一步探讨MBP-1分子高表达对食管癌细胞增殖和凋亡的影响以及以MBP-1分子为靶点的食管癌基因治疗等研究奠定了基础.

1 材料与方法 1.1 质粒载体和细胞株 质粒载体pcDNA3.1(+)由本研究室唐恩洁教授赠送.含MBP-1基因的质粒pCMV6-XLM购自Origene公司, 食管癌细胞株Eca109购自中国科学院上海细胞生物研究所. 1.2 主要试剂 PCR扩增试剂、T4噬菌体DNA连接酶、限制性内切酶HindⅢ和BamHⅠ、质粒提取试剂盒、脂质体转染试剂Lipofectin