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High-temperature-resistant mutant SOD with PTD and encoding gene and application thereof

A PTD-SOD-MUTANT, encoding gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of limited SOD application, poor thermal stability, and no specific receptors in cell membranes

Active Publication Date: 2020-03-10
SUZHOU UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical applications, SOD still has some shortcomings, such as poor thermal stability and easy inactivation at high temperature; there is no specific receptor on the cell membrane, and it is difficult to enter the cell to play a role; natural SOD is expensive to separate and purify, etc.
Although the current basic research has proved the efficacy and mode of action of SOD, due to the shortcomings of SOD in the prior art in terms of thermal stability and activity maintenance, the application of SOD in the fields of cosmetics, food and medicine has been greatly limited. , can not achieve the desired effect well

Method used

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  • High-temperature-resistant mutant SOD with PTD and encoding gene and application thereof
  • High-temperature-resistant mutant SOD with PTD and encoding gene and application thereof
  • High-temperature-resistant mutant SOD with PTD and encoding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0049] Expression and purification of PTD-SOD-MUTANT protein

[0050] The PTD-SOD-MUTANT gene sequence was synthesized by Sangon Bioengineering Shanghai (Co., Ltd.), and the prokaryotic expression vector was pet28b. Transform the recombinant plasmid into competent Escherichia coli BL21(DE3) by heat shock method, add isopropyl-β-D-thiogalactopyranoside (IPTG) for shaker culture, and induce PTD-SOD-MUTANT protein For expression, the shaker speed is 220rpm / min (rotation speed range 200-250rpm / min, preferably 220rpm / min), the induction temperature is 37°C (induction temperature range is 30-38°C, preferably 37°C), and the final concentration of IPTG is 1mM (IPTG The final concentration range is 0.1-1.5mM, preferably 1mM), and the induction time is 7 hours (the induction time range is 3-8h, preferably 7h). The bacteria after induced expression were treated by ultrasonic lysis, and the PTD-SOD-MUTANT protein was purified by adding Washing Buffer with an imidazole concentration of 25...

Embodiment 2

[0052] Inhibitory effect of PTD-SOD-MUTANT protein on mouse B16 melanoma cell proliferation in vitro

[0053] The effect of PTD-SOD-MUTANT protein and SOD protein (control) on the proliferation of mouse B16 melanoma cells was detected by MTT method. The principle of MTT method: succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-purple crystals and deposit them in cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve purple crystals in cells, and the light absorption value is measured at a wavelength of 570nm with a microplate reader, which can indirectly reflect the number of living cells.

[0054] The specific operation is as follows: take mouse B16 melanoma cells in the logarithmic growth phase, inoculate them into 96-well plates, and the number of cells per well is 1×10 3 ;Set the treatment groups for the two proteins according to three gradients of 0.2, 0.4, and 0.8 mg / mL, with 3 auxiliary...

Embodiment 3

[0057] In vivo therapeutic effect of PTD-SOD-MUTANT protein on malignant melanoma in mice

[0058] The mouse B16 melanoma cells in the logarithmic growth phase were taken, the culture fluid was aspirated, digested with trypsin and counted. Centrifuge at 1000rpm for 5 minutes, make a cell suspension with DMEM culture medium, and adjust the cell concentration to 1×10 6 individual / mL. Nine healthy purebred nude mice aged 4-6 weeks were injected with 0.1 mL of prepared tumor cell suspension in the left armpit, and repeated on the second day. The body weight of the nude mice was measured every 2 days, and the basic vital characteristics were observed. The growth of tumors in the axilla of the nude mice indicated that the modeling was successful. Every 2 days, the tumor diameter was measured with a vernier caliper. When the tumor diameter was about 1 cm, the animals were randomly divided into 3 groups, and 0.1 ml of PBS, PTD-SOD-MUTANT protein (concentration 0.8 mg / mL), SOD protei...

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Abstract

The invention discloses a PTD-SOD-MUTANT protein which is a fusion protein composed of a protein transduction structural domain PTD and SOD derived from thermophilic bacteria HB27, wherein I27L and D107A double mutation occurs in an SOD amino acid sequence. The PTD-SOD-MUTANT protein has the advantages of being good in thermal stability, high in activity and capable of penetrating through cell membranes to enter cells and the like. In-vitro and in-vivo experiments show that the protein has a very strong proliferation inhibition effect on mouse B16 melanoma cells and mouse malignant melanoma, and the protein and adriamycin (DOX) have a synergistic interaction effect on tumor cells. The PTD-SOD-MUTANT protein can be used as a pharmaceutical composition, a cosmetic active component, a food additive and the like, and has a great application value and wide application prospect in the fields of drug research and development, daily necessities, foods and the like.

Description

technical field [0001] The invention relates to an enzyme, its encoding gene and application, in particular to a high temperature-resistant mutant SOD with PTD, its encoding gene and application. Background technique [0002] Superoxide dismutase (SOD for short) is an enzyme containing metal ions discovered by American scientists Mccord and Fridovich in 1969 from bovine red blood cells. This enzyme catalyzes the generation of H from superoxide anion radicals 2 o 2 and O 2 , is an oxidoreductase that removes excess superoxide anion free radicals in organisms. According to the different metal ions in the active center of SOD, SOD can be divided into four types: Cu / Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD. SOD has a certain curative effect on cancer, inflammation, ischemia-reperfusion injury, radiation injury, etc., and can also reduce the toxic and side effects of anticancer drugs on cells and the heart. It is one of the most effective antioxidant enzymes with antitumor activity....

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70A61K38/44A61P35/00A61K8/66A61Q19/00A61Q19/02A61Q19/08A23L33/17
CPCC12N9/0089C12Y115/01001C12N15/70A23L33/17A61P35/00A61K8/66A61Q19/00A61Q19/02A61Q19/08A23V2002/00A61K38/00A23V2250/54
Inventor 韩宏岩许维岸林安安
Owner SUZHOU UNIV
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