Galectin-3重组真核表达载体的构建及其在NIH/3T3细胞中的表达

Construction of recombinant eukaryote expression vector encoding gene of galectin-3 and its expression in NIH/3T3 cells

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,并促进NIH/3T3细胞增殖。

Objective：To construct recombinant eukaryote expression vector containing Gal-3 gene and detect 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 the 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 restriction enzyme analysis and nucleic acid sequence successfully. The expression of Gal-3 in NIH/3T3 cells transient transfected by recombinant plasmid 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 of rats. Furthermore,our study found that Gal-3 protein could promote the proliferation of NIH/3T3 cells.