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A high temperature resistant mutant sod with ptd and its application

A PTD-SOD-MUTANT, mutant technology, applied in applications, medical preparations containing active ingredients, cosmetic preparations, etc., can solve the problem of expensive separation and purification of natural SOD, no specific receptors in cell membranes, limiting SOD application and other issues, to achieve the effect of great application value, great advantages and broad application prospects

Active Publication Date: 2022-07-22
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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  • A high temperature resistant mutant sod with ptd and its application
  • A high temperature resistant mutant sod with ptd and its application
  • A high temperature resistant mutant sod with ptd and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Expression and purification of PTD-SOD-MUTANT protein

[0028] The PTD-SOD-MUTANT gene sequence was synthesized by Sangon Bioengineering Shanghai (Co., Ltd.), and the prokaryotic expression vector was pet28b. The recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) by heat shock method, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added for shaking culture to induce PTD-SOD-MUTANT protein. Expression, shaker rotation speed is 220rpm / min (rotation speed range 200-250rpm / min, preferably 220rpm / min), induction temperature is 37 ℃ (induction temperature range 30-38 ℃, preferably 37 ℃), IPTG final concentration is 1mM (IPTG The final concentration range is 0.1-1.5 mM, preferably 1 mM), and the induction time is 7 hours (the induction time range is 3-8 h, preferably 7 h). The cells after induction and expression were treated by ultrasonic lysis, and the PTD-SOD-MUTANT protein was purified by Ni-NTA affinity chromatography column under natural c...

Embodiment 2

[0030] Inhibitory effect of PTD-SOD-MUTANT protein on the proliferation of mouse B16 melanoma cells in vitro

[0031] The effects of PTD-SOD-MUTANT protein and SOD protein (control) on the proliferation of mouse B16 melanoma cells were detected by MTT method. Principle of MTT method: Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-purple crystals and deposit in cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve purple crystals in cells, and the light absorption value is measured at 570nm wavelength with a microplate reader, which can indirectly reflect the number of living cells.

[0032] The specific operation is as follows: Take the mouse B16 melanoma cells in the logarithmic growth phase and inoculate them into a 96-well plate, and the number of cells in each well is 1×10 3 ; The two proteins were respectively set as treatment groups according to three gradients of 0.2, 0.4 and 0....

Embodiment 3

[0035] In vivo therapeutic effect of PTD-SOD-MUTANT protein on malignant melanoma in mice

[0036]The mouse B16 melanoma cells in the logarithmic growth phase were taken, the culture medium was aspirated, trypsinized and counted. Centrifuge at 1000 rpm for 5 minutes, make a cell suspension with DMEM medium, and adjust the cell concentration to 1 × 10 6 pcs / mL. Nine healthy purebred nude mice aged 4-6 weeks were taken, and 0.1 mL of the prepared tumor cell suspension was injected into the left armpit, which was 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 armpits of the nude mice marked the success of the modeling. The diameter of the tumor was measured with a vernier caliper every 2 days. When the diameter of the tumor 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) and S...

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Abstract

The invention discloses a PTD-SOD-MUTANT protein, which is a fusion protein composed of a protein transduction domain PTD and SOD derived from thermophilic bacteria HB27, wherein the SOD amino acid sequence has double mutations of I28L and D108A. PTD‑SOD‑MUTANT protein has the advantages of good thermal stability, high activity, and can enter cells through cell membranes. In vitro and in vivo experiments show that this protein has a strong inhibitory effect on the proliferation of mouse B16 melanoma cells and mouse malignant melanoma. The combination of this protein and doxorubicin (DOX) has a synergistic effect on tumor cells. PTD‑SOD‑MUTANT proteins can be used as pharmaceutical compositions, cosmetic active components, food additives, etc., and have great application value and broad application prospects in the field of drug research and development, daily necessities, and food.

Description

technical field [0001] The invention relates to enzymes and applications, in particular to a high temperature-resistant mutant SOD with PTD and applications. Background technique [0002] Superoxide dismutase (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. The enzyme catalyzes the generation of H from superoxide anion free radicals 2 O 2 and O 2 , is an oxidoreductase that scavenges excess superoxide anion free radicals in the body. According to the different metal ions in the active center of SOD, SOD can be divided into four categories: 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. It can also reduce the toxic and side effects of anti-cancer drugs on cells and heart. It is one of the most effective antioxidant enzymes with anti-tumor activity. Therefor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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