截断型突变体HBx-Mut120稳定表达真核细胞系的建立

Establishment of Truncated Mutant HBx-Mut120 Stably Expressing Eukaryotic Cell Lines

目的 成功构建HBx及其截断型突变体HBx-Mut120的真核载体,建立了两者稳定表达的HepG2细胞系,并研究了目的基因对细胞生长的影响。方法 采用PCR扩增HBx及其截断型突变体HBx-Mut120基因,然后分别克隆到PCMVTag2B载体中,并构建成重组质粒HBx-PCMV-Tag2B和HBx-Mut120-PCMV-Tag2B,经过PCR、酶切、测序鉴定重组质粒后,将其转染人肝癌细胞HepG2,经Game 418筛选后得到稳定表达目的基因的Hep G2细胞,最后通过RT-PCR和Western blot鉴定HBx、HBx-Mut120的mRNA和蛋白在HepG2细胞中的表达情况,并用流式细胞仪检测了各组细胞的周期。结果 (1)成功构建目的基因表达载体PCMV-Tag2B-HBx、PCMV-Tag2B-HBx-Mut120。(2)成功建立稳定表达HBx及其缺失片段的HBx蛋白的HepG2细胞系。(3)通过流式细胞仪检测各组细胞周期及对比,发现HepG2-HBx细胞和HepG2-HBx-Mut120细胞的生长分数(G_2+S)明显高于HepG2细胞及空载体转染的HepG2细胞。结论 本实验成功构建携带HBx及其截断型突变体HBxMut120基因的重组表达质粒,转染人肝癌细胞HepG2细胞后分别能够稳定表达HBx蛋白和截断型HBx蛋白,HBx及其突变体稳转细胞后更能促进HepG2细胞的增殖,且突变型目的基因稳转细胞系转移能力较前明显增强。为进一步研究截断型HBx突变体在肝癌的发生、发展中的作用奠定了实验基础。

Objective To construct the eukaryotic expression vector HBx truncated mutant HBx-Mut120 and establish HepG2 cell line with its stable expression,and research the effect of target gene on cell proliferation. Methods HBx and its truncated mutant HBx-Mut120 gene were amplified by PCR and then cloned into the PCMV-Tag2 B vectors to construct recombinant plasmids HBx-PCMV-Tag2 B and HBx-Mut120-PCMV-Tag2 B. the recombinant genes were confirmed by PCR,restriction enzyme digestion and DNA sequencing before they transfected human hepatoma cell line HepG2. We got HepG2 cells expressing stably target gene selected by Game418. Finally,the mRNA and protein expressions of HBx、HBx-Mut120 were determined by the reverse transcription-PCR and Western blotting. Cell cycles were analyzed by FCM flow cytometry. Results （1）The objective gene expression vectors PCMV-Tag2 B-HBx,PCMV-Tag2 B-HBx-Mut120 was constructed successfully.（2） The HepG2 cell line stably expressing HBx and delected protein—HBx-Mut120 was successfully established.（3） Compared these cell cycles tested by cytometry instrument,we found that growth points（G_2＋ S） of HepG2-HBx and HepG2-HBx-Mut120 were significantly higher than of HepG2 and HepG2 with empty vector transfection. Conclusion The HBx and the truncated mutant HBx-Mut120 gene recombinant plasmid were successfully constructed in the experiment,which transfected human hepatocellular carcinoma HepG2 cells and are capable of expressing HBx protein and the truncated protein stability. That provides the experimental basis for further studying the role of truncated HBx mutants in the occurrence and development of HCC.