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Construction of domestic porcine tumor necrosis factor mutant and protein expression purification method

A technology of tumor necrosis factor and mutants, applied in the direction of tumor necrosis factor and peptide preparation methods, chemical instruments and methods, etc.

Inactive Publication Date: 2015-04-29
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Tumor necrosis factor (TNF-α), as the cytokine with the best anti-tumor effect found so far, has always been a research hotspot at home and abroad, but almost all researches are around human TNF-α, and rarely involve domestic animals. Tumor necrosis factor

Method used

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  • Construction of domestic porcine tumor necrosis factor mutant and protein expression purification method
  • Construction of domestic porcine tumor necrosis factor mutant and protein expression purification method
  • Construction of domestic porcine tumor necrosis factor mutant and protein expression purification method

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Experimental program
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Effect test

Embodiment 1

[0031] The construction of porcine tumor necrosis factor mutant (PmTNF-α) gene, the pig spleen was obtained from Nanjing Qixia Yaohua pig slaughterhouse, and it was Landrace pig:

[0032] (1) Total RNA extraction: RNA extraction kit (TIANGEN) was used to extract total RNA from porcine spleen tissue according to its operation manual, and its quality and purity were identified by formaldehyde-denatured agarose gel electrophoresis, and its concentration was determined by an ultraviolet spectrophotometer. SMART TM RACE kit (TaKaRa) was reverse transcribed into first-strand cDNA.

[0033] (2) Design large fragment primers containing mutation sites, and construct tumor necrosis factor mutant genes by PCR method. Primer sequences such as SEQ ID NO.4 and SEQ ID NO.5, using the cDNA template obtained by reverse transcription for PCR:

[0034]①The reaction system is 50 μl, 10 μmol / L F1, 2 μl each of R1, 2.5 mmol / L dNTP 8 μl, 2×GC buffer II 25 μl, cDNA template 3 μl, DreamTaq enzyme 0...

Embodiment 2

[0037] Construction of recombinant expression vector of porcine tumor necrosis factor (TNF-α) mutant, its induced expression in Escherichia coli, purification and identification;

[0038] According to the constructed TNF-α mutant sequence (gene sequence such as SEQ ID NO.2), design primers F2 and R2. The 5' end of F2 has a BamHI restriction site, and the 5' end of R2 has a HindⅢ restriction site. The first-strand cDNA of porcine is used as a template, and F2 and R2 are used as primers (F2 5'–CGCGGATCCAAGCGCAAGCCCGTCGCCCACGTTGT-3' (SEQ ID NO.6), R25'-CCCAAGCTTTCAGAAGGCAATGATCCCAAAATAGT-3' (SEQ ID NO.7), the gene was amplified by PCR (10×pfu Buffer 5 μl, dNTP (2.5mM) 4 μl, F2 (10 μmol / L) 2 μl, R2 (10 μmol / L) 2μl, H 2 O 33.5 μl, cDNA 3 μl, pfu Taq 0.5 μl) The reaction conditions are as follows: 94 ° C for 5 min, (94 ° C for 30 s, 59 ° C for 30 s, 72 ° C for 1 min), 35 cycles, and finally 72 ° C for 7 min, and then the PCR product was tapped and recovered , the recovered produc...

Embodiment 3

[0044] Analysis of His by CCK-8 method 6 - Cytotoxic activity of PmTNF-α on L929 cells;

[0045] Culture L929 cells in 7ml 1640 (10% FBS) medium for 24 hours. After removing the medium, digest 1ml of trypsin for 30s, add 1ml of medium to stop the digestion, resuspend the cells, pipette into a 15ml centrifuge tube, 1000g, 20°C, 3min. After removing the supernatant, add 2ml of medium (1640, 10% FBS), resuspend the cells, pipette 1ml into a 50ml centrifuge tube, add 10% FBS 1640 medium to dilute, and count on a counting plate to make the number of cells 3×10 5 pieces / ml. Pipette the above-mentioned diluted cell suspension into a 96-well plate, 100 μl per well, 37°C, 5% CO 2 , cultivated for 24h. Remove culture medium. Use 10% FBS 1640 medium to configure different concentrations of TNF-α dilutions (divided into two groups of wild-type and mutant types), and then add 100 μl to each well to make the final concentration of TNF-α reach 10 5 、10 4 、10 3 、10 2 , 10, 10 0 、10 ...

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Abstract

The invention relates to site-directed mutagenesis of a domestic porcine tumor necrosis factor (TNF-alpha) gene, and a protein preparation method and application. One mutant gene of the porcine tumor necrosis factor (TNF-alpha) has the sequence disclosed as SEQ ID NO.2. The mutant gene translation protein amino acid sequence is disclosed as SEQ ID NO.3; the mutant can be connected into a pET28a prokaryotic expression plasmid and transformed into Escherichia coli BL21 (DE3) to induce expression, and Ni<+>- / NTA column purification is performed to obtain the recombinant His6-PmTNF-alpha; and the CCK-8, LDH detection and other experiments prove that the mutant protein can obviously enhance the cytotoxicity for L929 cells and lower the toxic or side effects on normal cells L02 as compared with the TNF-alpha wild type protein.

Description

technical field [0001] The invention relates to the field of biogenetic engineering, in particular to the technology of construction, expression, protein purification and activity identification of pig tumor necrosis factor mutant gene. Background technique [0002] Tumor necrosis factor (tumor necrosis factor, TNF-α) is a cytokine discovered in 1975 that has anti-tumor effects. In addition to directly inhibiting the proliferation and cell necrosis of tumor cells, TNF-α also affects the growth and differentiation of other cells (including cardiomyocytes), and at the same time resists viruses and bacterial infections; stimulates T cells, B cells, monocytes Macrophages, etc., to enhance their functions; induce inflammatory responses, promote the production and secretion of IL1, IL2, IL6, etc.; promote the expression of EGFR and major histocompatibility antigen type II antigen (MHCⅡAg), etc., in the host defense response play an important role in. TNF-α mainly realizes its bi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/28C07K14/525C12N15/63C07K1/36C07K1/34C07K1/16A61K38/19A61K39/39A61P37/04
Inventor 张双全朱善元杨林赵东伟
Owner NANJING NORMAL UNIVERSITY
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