材料和方法.doc

Galectin-3重组真核表达载体的构建及其在NIH/3T3细胞中的表达 胡圳圳 蒋秀琴 高才杰 郑大同* （南京医科大学第二附属医院中心实验室 江苏 南京 210011） [摘 要] 目的：构建半乳糖凝集素-3 （Gal-3） 的真核表达载体，在NIH/3T3细胞中表达，并检测其表达。方法：通过DNA重组技术和PCR方法从人肿瘤细胞克隆Gal-3基因，插入真核表达载体pEGFP-N1中，通过酶切和测序鉴定重组载体的正确性；采用脂质体转染技术将重组质粒pEGFP-Gal-3瞬时转染NIH/3T3细胞，经荧光和Western blot方法检测Gal-3表达，MTT法检测Gal-3对NIH/3T3细胞增殖的影响。结果：限制性内切酶鉴定和核酸序列测序证实成功构建含Gal-3的重组真核表达载体pEGFP-Gal-3。以重组质粒瞬时转染NIH/3T3细胞，检测到Gal-3蛋白表达，并证实Gal-3蛋白促进NIH/3T3细胞增殖。结论：成功构建的pEGFP-Gal-3真核表达载体在小鼠NIH/3T3细胞中成功表达，其促进NIH/3T3细胞增殖。 [关键词] 半乳糖凝集素-3；NIH/3T3细胞；细胞增殖 [中图分类号] Q786 [文标识码] A [文章编号] Construction of the recombinant eukaryote expression vector encoding gene of galectin-3 and its expression in NIH/3T3 cells HU Zhen-zhen, JIANG Xiu-qin, Gao Cai-jie, ZHENG Da-tong* (Center Laboratory, Second Affiliated Hospital of NJMU, Nanjing 210011, China) [Abstract] Objective: To construct the recombinant eukaryote expression vector containing Gal-3 gene and detect the protein expression in NIH/3T3 cells. Methods: Gal-3 gene was obtained by PCR from cancer cells. PCR product of Gal-3 was then clone into the eukaryote expression vector pEGFP-N1, then the recombinant vector pEGFP-Gal-3 was transfected into the NIH/3T3 cells. After transfection of the recombinant vector, the protein expression of Gal-3 in NIH/3T3 cells was detected by Western blot. The effect of Gal-3 on cell proliferation was investigated by MTT assay. Results: The recombinant eukaryote expression vector encoding Gal-3 was constructed successfully. The expression of Gal-3 in NIH/3T3 cells could be detected by Western blot. In addition, Gal-3 protein could promote the proliferation of NIH/3T3 cells. Conclusion: The new recombinant expression vector pEGFP-Gal-3 was constructed and expressed successfully in NIH/3T3 cells. Furthermore, our study found that Gal-3 [Key words] Galectin-3; NIH/3T3 cell; Cell proliferation [基金项目] 南京医科大学科技发展基金重点项目（2012NJMU088）。 *通讯作者，Email：dtzheng@ 半乳糖凝集素-3（Gal-3），属于半乳糖凝集素家族（Galectin family）成员，广泛表达于正常组织和