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目的构建DNA甲基转移酶3B(DNMT3B)的一种异构体3B7的真核质粒表达载体,在体外评价其对人胚肾细胞株293A增殖影响。方法据GenBank中人DNMT3B7 cDNA序列设计并合成特异性引物,以DNMT3B1为模板,利用高保真Taq酶,进行PCR技术扩增DNMT3B7,并将扩增产物克隆到真核表达载体pCMV-2B中。将携带DNMT3B7基因的质粒pCMV-DNMT3B7及空质粒pCMV-2B转染293A细胞,通过G418筛选出稳定表达的细胞系,MTT法检测细胞的增殖;流式细胞仪检测细胞的细胞周期分布。同时检测p21蛋白表达情况。结果成功构建DNMT3B7的真核表达载体并筛选出稳定表达株,与转染空质粒组相比,稳定过表达DNMT3B7组293A细胞株增殖减慢(P<0.01);过表达DNMT3B7的293A细胞S期细胞比例显著增多(P<0.05)。p21 mRNA和蛋白表达增加(P<0.05)。结论 DNMT3B7基因过表达可抑制293A细胞增殖,p21可能参与了这一过程的调节。
Objective To construct eukaryotic plasmid expression vector 3B7, an isoform of DNA methyltransferase 3B (DNMT3B), and evaluate its effect on the proliferation of human embryonic kidney cell line 293A in vitro. Methods According to the DNMT3B7 cDNA sequence in GenBank, specific primers were designed and synthesized. DNMT3B1 was used as template to amplify DNMT3B7 by high-fidelity Taq polymerase. The amplified product was cloned into eukaryotic expression vector pCMV-2B. 293T cells were transfected with plasmid pCMV-DNMT3B7 carrying DNMT3B7 gene and empty plasmid pCMV-2B. The stable cell lines were selected by G418. Cell proliferation was detected by MTT assay. The cell cycle distribution was analyzed by flow cytometry. Simultaneous detection of p21 protein expression. Results The DNMT3B7 eukaryotic expression vector was successfully constructed and the stable expression strains were screened out. The proliferation of 293A cells stably over-expressing DNMT3B7 group was slower than that of transfected DNMT3B7 group (P <0.01) The proportion of cells increased significantly (P <0.05). p21 mRNA and protein expression increased (P <0.05). Conclusion DNMT3B7 gene overexpression can inhibit 293A cell proliferation, p21 may be involved in the regulation of this process.