[Research on construction and expression of recombinant eukaryotic expression plasmid for mouse adiponectin].

ABSTRACT

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.