TIMP-3基因真核表达载体的构建及其对乳腺癌MDA-MB-453细胞生长及侵袭的抑制作用

doi:10.13481/j.1671-587x.20160118

Abstract

Abstract:

Objective: To construct a mammalian tissue inhibitor of metalloproteinase-3 (TIMP-3)recombinant eukaryotic expression vecor and transfect the breast cancer MDA-MB-453 cells,and to explore the inhibitory effect of TIMP-3 gene on the growth and invasion of MDA-MB-453.Methods: The TIMP-3 cDNA was obtained from the human fresh placenta by RT-PCR. TIMP-3 gene was connected to pMD18-T vector.The recombinant pMD18-T vector and pcDNA3.1 were digested by EcoR Ⅰ and XbaⅠ. Then TIMP-3 gene was subcloned into pcDNA3.1 vetor to construct pcDNA-TIMP-3 recombinant vector and enzyme digestion identification and sequencing.The MDA-MB-453 cells were divided into normal control group,pcDNA group (transferted with pcDNA)and pcDNA-TIMP-3 group (transfected with pcDNA3.1-TIMP-3). The proliferation activities and invasion abilities of the MDA-453 cells in various groups were determined by MTT method and Boyden invasion experiment. Results: The correct construction of pcDNA-TIMP-3 was identified by means of restriction enzyme EcoRⅠ and XbaⅠ. The gene fragment of 633 bp and linearized vector were obtained.The result of TIMP-3 gene sequencing was correct.The Western blotting results showed that the transfected MDA-MB-453 cells expressed TIMP-3. The proliferation activity and invasion ability of MDA-MB-453 cells in pcDNA-TIMP-3 group were reduced significantly compared with normal control group and pcDNA group(P<0.05). Conclusion: The pcDNA-TIMP-3 eukaryotic expression vector is constructed successfully and TIMP-3 can be expressed in the MDA-MB-453 cells.The pcDNA-TIMP-3 has inhibitory effect on the growth and invasion of MDA-MB-453 cell lines.

Key words: tissue inhibitor of metalloproteinase-3, eukaryotic expression vector, MDA-MB-453 cell line

CLC Number:

R737.9

WANG Dan, WANG Zhenyu, SHI Guang, YIN Guanghao. Construction of eukaryotic expression vector of TIMP-3 gene and its inhibitory effect on growth and invasion of MDA-MB-453 cells[J].Journal of Jilin University Medicine Edition, 2016, 42(01): 89-93.

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Construction of eukaryotic expression vector of TIMP-3 gene and its inhibitory effect on growth and invasion of MDA-MB-453 cells

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