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G6PD over-expressed ACHN stable cell strain and construction method thereof

A construction method and cell line technology, applied in the field of medical molecular biology

Inactive Publication Date: 2015-04-22
KUNMING MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But what is the relationship between the expression or activity of G6PD and the occurrence and development of renal cell carcinoma and its mechanism? At present, there are no relevant reports at home and abroad

Method used

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  • G6PD over-expressed ACHN stable cell strain and construction method thereof
  • G6PD over-expressed ACHN stable cell strain and construction method thereof
  • G6PD over-expressed ACHN stable cell strain and construction method thereof

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Embodiment Construction

[0033] 1. Cloning of G6PD gene

[0034] Use the Primer 5.0 software to design the PCR forward and reverse primers for human G6PD gene (NM_001042351.2) cDNA. The forward primer 5'-GGAATTCATGGCAGAGCAGGTGGCCCTG-3' introduces the EcoR Ⅰ restriction site (G↓AATTC), and the reverse The primer 5'-ACGCGTCGACTCAGAGCTTGTGGGGGTTCAC-3' introduced a Sal I restriction site (G↓TCGAC). Using the pMD19-Tsimple-G6PD-WT plasmid DNA previously constructed by our research group as a template, the cDNA fragment containing G6PD was amplified by PCR, with a length of 1548bp. The PCR reaction system (50 μL) is as follows:

[0035]

[0036] After mixing, perform PCR amplification. The PCR cycle parameters are: 94°C for 10 min→94°C for 1 min→55°C for 45s→72°C for 1 min, and after 28 cycles, extend at 72°C for 10 min. Take 3 μL of PCR products for 1% agarose gel electrophoresis detection. The result is as figure 1 As shown, using the G6PD forward and reverse primers, using the pMD19-Tsimple-G6PD-...

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Abstract

The invention relates to a related technology of medical molecular biology, and in particular relates to a cell research model constructed by adopting a siRNA jamming technology. A G6PD over-expressed ACHN stable cell strain disclosed by the invention is a G6PD over-expressed ACHN stable cell strain. The cell strain is obtained by pressurizing and screening a pBABE-puro-G6PD transfected human renal cell adenocarcinoma cell strain ACHN through puromycin, verifying Real-time PCR and Western blot, carrying out repeated passage and cryopreservation resuscitation. The G6PD expressed quantity of the obtained cell strain is increased by above 40 times. A construction method disclosed by the invention comprises the following steps of: 1, cloning a G6PD gene; 2, constructing a pBABE-puro-G6PD recombinant expressed vector; 3, carrying out transfection on an ACHN cell strain; and 4, screening and identifying an ACHN-G6PD cell strain. Construction of the stable cell strain provides a cell model for researching the relevance between G6PD and renal cell carcinoma and relevance mechanism and also can lay a foundation for generation and development mechanism of renal cell carcinoma, and discovery of a novel drug action target.

Description

technical field [0001] The invention relates to related technologies of medical molecular biology, in particular to constructing cell research models by using siRNA interference technology. Background technique [0002] Glucose-6-phosphate dehydrogenase (G6PD) widely exists in various tissue cells of organisms and is a key enzyme of the pentose-phosphate pathway (PPP). The G6PD gene is located at Xq2.8 (NM_000402), with a full length of 18kb, consisting of 13 exons and 12 introns. The full length of its mRNA is 2395bp, the cDNA is 1548bp, encoding 515 amino acids, and the molecular weight of a single subunit is 59kDa , the active G6PD exists in dimer or tetramer state. G6PD catalyzes the dehydrogenation of glucose-6-phosphate (glucose-6-phophte, G-6-P) to generate 6-phosphogluconolactone, and at the same time produces reduced nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate, NADPH). NADPH can maintain intracellular reduced glutathio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/53
Inventor 朱月春张巧王艳玲况应敏狄勇李玉倩杨惠鑫
Owner KUNMING MEDICAL UNIVERSITY
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