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Mbr-FPGS (folylpolyglutamate synthetase) efficient expression vector and construction method and application thereof

A high-efficiency expression, mbr-fpgs technology, applied in the field of FPGS high-efficiency expression vectors and construction, to achieve the effects of enhancing sensitivity, good economic benefits and social effects, and improving protein expression levels

Inactive Publication Date: 2012-02-01
NANJING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, while MTX inhibits DNA synthesis in tumor cells, it also inhibits DNA synthesis in normal cells, leading to a variety of adverse reactions.

Method used

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  • Mbr-FPGS (folylpolyglutamate synthetase) efficient expression vector and construction method and application thereof
  • Mbr-FPGS (folylpolyglutamate synthetase) efficient expression vector and construction method and application thereof
  • Mbr-FPGS (folylpolyglutamate synthetase) efficient expression vector and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Total RNA was extracted from Jurkat cells by the Trizol method, and the steps included: (1) Take about 10 6For each Jurkat cell, after adding 1 ml Trizol, blow and lyse the cells with a 1 ml pipette, then transfer the cell lysate to a 1.5 ml centrifuge tube, and place at room temperature for 5 min. (2) Add 0.2 ml of chloroform, shake vigorously for 15 s, let stand at room temperature for 2-3 min, then centrifuge at 12,000 g for 15 min at 4°C. (3) Transfer the upper aqueous phase to a new 1.5 ml centrifuge tube, add 0.5 ml isopropanol, mix well, let stand at room temperature for 10 min, and centrifuge at 12,000 g for 10 min at 4°C. (4) Discard the supernatant, add 1 ml of 75% ethanol, wash, and centrifuge the above sample at 4°C, 7,500 g for 8 min. (5) Carefully discard the supernatant, dry at room temperature or in vacuum for 5-10 min, add 20 μl RNase-free water to dissolve. (6) RNA quantification and purity analysis were carried out with a UV spectrophotometer. The r...

Embodiment 2

[0049] The human BCL2 gene is a proto-oncogene cloned from follicular-like B-cell lymphoma with t(14,18) chromosome translocation by Tsujimoto et al. in 1985 [SetoM, JaegerU, HockettRD, GraningerW, BennettS, GoldmanP, KorsmeyerSJ: Alternativepromotersandexons ,somatic mutation and deregulation of the Bcl-2-Igfusion gene in lymphoma.EMBOJ1988,7(1):123-131], the encoded product BCL2 protein is a key factor regulating apoptosis [HockenbergyD, NunezG, MillimanC, SchreiberRD, KorsmeyerRD, KorsmeyerSJ: Bcl-2isaninnermitochondrialmembraneprotein9Nblocks4,3pro (6299):334-336.], Chromosome 14 and 18 translocations are the most common chromosomal translocations associated with human cancer. Most of the translocations in the lymphoma cells of patients with follicular lymphoma occurred in the major breakpoint region (mbr) of the 3'-untranslated region (3'-UTR) of the BCL2 gene. Using a reporter gene system and gene-directed targeting technology, it has been confirmed that the mbr sequence...

Embodiment 3

[0055] The MTT method was used to detect the half inhibitory concentration (IC50) of MTX. The method was as follows: the Jurkat cells in the logarithmic growth phase were collected and seeded in a 96-well plate. According to the peak plasma concentration of clinical drug MTX, 5 drug concentration gradients were prepared, and the concentration was: 2×10 -6mg / ml, 1×10 -5 mg / ml, 5×10 -5 mg / ml, 2.5×10 -4 mg / ml, 1.25×10 -3 mg / ml; dissolved in RPMI-1640 medium containing 10% calf serum. 10,000 Jurkat cells were inoculated per well, and different concentrations of MTX were added. Three replicate wells were set up for each concentration, and cultured for 24h, 36h, 48h, 60h, and 72h. Add 5mg / ml MTT10ul, incubate for 4h, add 100ul triple solution (10% SDS, 5% isobutanol, 0.012mol / L HCl, dissolved in distilled water), and measure the absorbance value (A) at 570nM wavelength on an automatic microplate reader. Apply SPSS10.0 software to calculate IC50. Cell viability = (test well A v...

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Abstract

The invention discloses an mbr-FPGS (folylpolyglutamate synthetase) efficient expression vector. In the vector, a plasmid vector is pEGFP-C1, an mbr sequence with the length of 279bp is contained on the upstream of a cytomegalovirus (CMV) promoter of the pEGFP-C1, and a protein coding region of FPGS is contained between two restriction enzyme cutting sites, namely Hind III and EcoR I of the pEGFP-C1; and the mbr sequence is shown in a sequence table 2. An IC50 result of the constructed mbr-FPGS efficient expression plasmid which is measured by a Western Blot experiment and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) experiment proves that: the FPGS expression level of the mbr-FPGS efficient expression plasmid is improved by 30 percent (p is less than 0.05) compared with that of an FPGS comparison plasmid, the mbr-FPGS efficient expression plasmid can obviously improve the sensitivity of cancer cells to methotrexate (MTX) and has a certain dose-time dependence,and the IC50 value (72h) of an mbr-FPGS efficient expression group cell is 2.63ng / ml which is one seventh (p is less than 0.05) of the IC50 value (19.01ng / ml) of a comparison group cell approximately.

Description

technical field [0001] The invention belongs to the technical field of gene transcription regulation in genetic engineering. It specifically relates to an FPGS high-efficiency expression vector, a construction method and an application. Background technique [0002] Since Farber et al first reported in 1948 that the precursor of methotrexate, aminopterate, could bring remission to children with acute leukemia, chemotherapy based on this has emerged. Methotrexate (Methotrexate, MTX) has become the most widely used anti-metabolite drug for leukemia, lymphoma, head and neck tumors, osteosarcoma and various autoimmune diseases. Studies have shown that MTX is a folic acid congener, which mainly enters cells with the help of reduced folic acid carrier (RFC), and the MTX entering cells forms methotrexate polyglutamate under the action of folyl polyglutamate synthase (FPGS) MTXPG and MTXγ-glutamate hydrolase (GGH) act to hydrolyze MTXPG, and the dynamic balance between the two kee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66A61K48/00A61P35/00A61P35/02A61K31/519
CPCY02A50/30
Inventor 孙玉洁胡文佳韩晓
Owner NANJING MEDICAL UNIV
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