Jing Xiao-Mei, Wen Yan-jun, Jia Yong-xi, Wang Yong-sheng, Li Jiong, Kan Bing, Deng Hong-xin
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2008 Jul;39(4):531-4.
To construct the recombinant eukaryotic expression plasmid for mouse adiponectin so as to supply experimental basis for research further on the function of adiponectin.
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.
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.
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.
构建小鼠脂联素重组真核表达质粒，为进一步研究脂联素的功能提供实验依据。
从小鼠脂肪组织中提取总RNA，应用逆转录聚合酶链反应（RT-PCR）扩增小鼠脂联素cDNA的完整编码区，并将扩增产物与pCDNA3.1(+)载体连接；然后通过测序验证重组质粒，并检测其在体外的重组表达及对小鼠乳腺癌细胞4T1的细胞增殖抑制作用。
PCR扩增重组质粒后得到一个744 bp的片段，经测序及蛋白质免疫印迹法（Western-blot）检测表达予以证实。此外，与体外对照试验相比，重组质粒转染细胞48 h后，可观察到4T1细胞有明显的形态学变化，细胞皱缩或塌陷，细胞增殖受到抑制。在流式细胞术分析中，也可观察到转染的4T1细胞凋亡比例明显高于对照。
成功构建了小鼠脂联素重组真核表达质粒，其对小鼠乳腺癌细胞4T1的增殖有明显抑制作用。
