DLD1 stable cell strain efficiently expressing human PKM1 protein and construction method thereof

A high-efficiency expression and construction method technology, applied in the direction of microorganism-based methods, cells modified by introducing foreign genetic material, botany equipment and methods, etc., can solve the problems of no persistence, and achieve stable expression and transfection efficiency High, low dosage effect

Inactive Publication Date: 2013-06-19
SHANXI UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the prior art, there is no colon cancer DLD1 stable cel

Method used

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  • DLD1 stable cell strain efficiently expressing human PKM1 protein and construction method thereof
  • DLD1 stable cell strain efficiently expressing human PKM1 protein and construction method thereof
  • DLD1 stable cell strain efficiently expressing human PKM1 protein and construction method thereof

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Experimental program
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Embodiment 1

[0019] 1. Construction of recombinant human pyruvate kinase PKM1 gene lentiviral expression vector

[0020] According to the coding sequence of PKM1 gene (GenBank NM_182471.2), specific primers with BamHI and XhoI restriction sites and protective bases at both ends were designed: 5'-CCGCTCGAGGCCACCATGTCGAAGCCCCATAGTGAAG-3' and 5'-CGCGGATCCCGCGGCACAGGAACAACACG-3', from human The PKM1 gene was amplified in the cDNA of colon cell NCM460. BamHI and XhoI double enzyme digestion, gel recovery of the target gene fragment, and the same BamHI and XhoI double enzyme digestion pLVX-AcGFP1-N1 vector, to obtain the recombinant plasmid pLVX-AcGFP1-N1-PKM1, identified by double enzyme digestion (see figure 1 ) and sequence identification of recombinant plasmids (see figure 2 ).

[0021] 2. Culture and passage of DLD1 cells and 293T cells

[0022] The cells came from the Institute of Biotechnology of Shanxi University. The revived DLD1 cells were placed in a 10cm culture dish and cultured...

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Abstract

The invention discloses a DLD1 stable cell strain efficiently expressing human PKM1 protein and a construction method thereof, and belongs to the field of animal cell strains. The construction method comprises the following steps of: constructing a recombined human PKM1 gene lentiviral expression vector; packaging and harvesting lentivirus; transfecting DLD1 cell; and obtaining the stable cell strain through puromycin screening, RT-PCR and Western Blot technological detection. The stable cell strain constructed according to the method provides a powerful tool for searching colon cancer Wnt signal path and tumor energy metabolism.

Description

technical field [0001] The invention relates to a DLD1 stable cell strain highly expressing human pyruvate kinase PKM1 protein and a construction method thereof. Background technique [0002] Even in the presence of oxygen, tumor cells are primarily metabolized by glycolysis rather than oxidative phosphorylation, accompanied by increased rates of glucose consumption and lactate production. This abnormal phenomenon of glucose metabolism is called the Warburg effect. Glycolysis, as the main production method of tumor cells, plays an important role in the survival and proliferation of tumor cells. Why does the way tumor cells produce energy change? Although this process is not fully understood, pyruvate kinase is now thought to play an important role in the alteration of energy metabolism in tumor cells. [0003] Pyruvate kinase is a key and rate-limiting enzyme in the glycolytic pathway. In mammalian cell lines, there are four isozymes of pyruvate kinase, PKM1, PKM2, PKL a...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/54C12N15/867C12R1/91
Inventor 杨鹏李卓玉李宗伟付荣李汉卿
Owner SHANXI UNIV
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