Adiponectin真核表达质粒的构建与表达研究

目的构建重组鼠adiponectin真核表达质粒,为进一步研究adiponectin的功能提供实验基础。方法提取鼠脂肪组织总RNA,运用RT-PCR方法扩增鼠adiponectincDNA完全编码区,将扩增产物连接至pCDNA3.1( )载体,对重组质粒进行测序验证,并检测其体外表达和对小鼠乳腺癌细胞4T1的增殖抑制作用。结果重组真核表达质粒经PCR扩增后获得一744bp片段,并经测序证实,Western-blot检测到其体外表达。在体外的细胞学实验中,将重组质粒转染入小鼠乳腺癌细胞4T148h后,与对照组相比,可以观察到明显的细胞形态学改变,细胞体积变小皱缩,4T1细胞生长明显抑制。流式细胞术检测到该组细胞中凋亡细胞所占比例明显高于对照组。结论成功构建了鼠重组adiponectin真核表达质粒,该质粒对小鼠乳腺癌细胞4T1的增殖有明显的抑制作用。

Research on Construction and Expression of Recombinant Eukaryotic Expression Plasmid for Mouse Adiponectin

Objective To construct the recombinant eukaryotic expression plasmid for mouse adiponectin so as to supply experimental basis for research further on the function of adiponectin.Methods Extracting the total RNA from mouse adipose tissue,applying RT-PCR to amplify the complete coding region of mouse adiponectin cDNA and connecting the amplified product with pCDNA3.1( )vector;then verifying the recombinant plasmid by sequencing and detecting in vitro recombinant expression and cellular proliferation depression to mouse breast cancer cell 4T1.Results A 744 bp fragment was got after PCR amplification with the recombinant plasmid,which was confirmed by sequence and expression detected by Western-blot.Besides,compared with the control in vitro test,visible morphological changes of 4T1 cell could be observed after recombinant plasmid cell transfection for 48 h,with cells collapsed or shrunk and cellular proliferation inhibited.During the flow cytometry analysis,the transfected 4T1 cells could also be observed obviously to have a greater proportion of cell apoptosis than the control could be detected.Conclusion The recombinant eukaryotic expression plasmid for mouse adiponectin is successfully constructed,which has an obvious inhibition effect on the proliferation of mouse breast cancer cell 4T1.