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Fusion protein GST-SOD (superoxide dismutase)1-X-R9 as well as preparation method and application thereof

A GST-SOD1-X-R9, 1. GST-SOD1-X-R9 technology, applied in the field of fusion proteins, can solve problems such as application limitations, and achieve the effects of protecting against radiation damage, reducing free radical damage, and preventing radiation damage

Active Publication Date: 2017-11-28
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for all radioprotectants, a question that cannot be ignored is whether the protective agent can also protect tumor tissues while protecting normal tissues from radiation damage, thereby affecting the effect of radiotherapy
Well-known small molecule antioxidants such as vitamin E can reduce the damage of normal tissues caused by radiation, but they also have a strong protective effect on tumor tissues, which limits their application in radiation protection.

Method used

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  • Fusion protein GST-SOD (superoxide dismutase)1-X-R9 as well as preparation method and application thereof
  • Fusion protein GST-SOD (superoxide dismutase)1-X-R9 as well as preparation method and application thereof
  • Fusion protein GST-SOD (superoxide dismutase)1-X-R9 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Construction Features of GST-SOD1-X-R9

[0051] (1) Construction of GST-SOD1-X-R9

[0052] The whole gene sequence of SOD1-X-R9 was synthesized by Sangon Bioengineering Shanghai (Co., Ltd.), and EcoRI and XhoI restriction sites were introduced into its upstream and downstream respectively, and it was combined with the pGEX-4T-1 plasmid (full sequence Containing the GST sequence in SOD1-X-R9 and pGEX-4T-1 were digested with EcoRI and XhoI, and then the SOD1-X-R9 and pGEX-4T-1 were ligated overnight at 4°C with T4 DNA ligase and transformed into Escherichia coli For BL21(DE3), single colonies were picked and cultured, and the plasmids were extracted for EcoRI and XhoI double enzyme digestion identification, and the positive clones identified correctly by EcoRI and XhoI were identified by DNA sequencing by Sangon Bioengineering Shanghai (Co., Ltd.).

[0053] (2) Purification and identification of GST-SOD1-X-R9

[0054] The high-purity GST-SOD1-X-R9 fusion p...

Embodiment 2

[0071] Example 2: Protective effect on radiation injury in normal mice

[0072] 1. The mice were fed normally for three days before irradiation, and they were free to eat and drink. Will be randomly divided into 10 groups (n=5 n=5): normal group (CON), radiation control group (XRT); amifostine group (AMFT+XRT); GST-SOD1-R9, GST-SOD1-X - experimental groups of R9 fusion proteins;

[0073] Normal group (CON): simple intraperitoneal injection of 0.5 mL of normal saline without radiation;

[0074] Radiation-only group (XRT): Before radiation, 0.5 mL of normal saline was injected into the abdominal cavity for irradiation;

[0075] Amifostine group (AMFT+XRT): 0.5 mL of amifostine was injected intraperitoneally at a dose of 200 mg / kg body weight 0.5 h before radiation;

[0076] Experimental group (GST-SOD1-R9+XRT, GST-SOD1-X-R9+XRT): 0.5 mL of the corresponding fusion protein was injected 2 hours before radiation, divided into three doses: 0.5 mg / mL, 0.75 mg / mL, 1.5 mg / mL. GS1...

Embodiment 3

[0100] Example 3: Effects on tumors in tumor-bearing mice after radiation

[0101] (1) Tumor formation by subcutaneous injection:

[0102] Cell culture: Hep G2 cells were placed in DMEM medium containing 10% newborn bovine serum and 1% double antibody in saturated humidity and 5% CO 2、Cultivate at 37°C, when the cells grow to 70-80% confluence, trypsinize and subculture;

[0103] Subcutaneous planting of cells: When the number of cultured cells reaches a certain level, wash twice with serum-free cell culture medium, trypsinize to collect cells, and use a cell counting plate for cell counting. Collect the cells at 1000rpm for 5min, dilute the collected cells to 2×10 with serum-free cell culture medium 7 cells / ml, add Matrigel at a ratio of 1:1 and mix well, then quickly inject 100 μl / only of the cell suspension under the armpits of nude mice (about 20 g), and the cell suspension must be mixed before each injection. All experimental mice were reared in a laminar flow rack wit...

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Abstract

The invention relates to the field of fusion protein, and particularly provides fusion protein GST-SOD (superoxide dismutase)1-X-R9 as well as a preparation method and an application thereof. A linker peptide X and cell-penetrating peptide R9 are fused with a gene recombination technology according to the gene sequence of copper-zinc SOD in Genbank, SOD1-X-R9 is obtained, the synthesized SOD1-X-R9 is inserted into a pGEX4T-1 plasmid containing a GST sequence, pGEX4T-1-SOD1-X-R9 expression plasmid is obtained, and soluble protein GSTSOD1-X-R9 with high expression quantity is obtained through expression in Escherichia coli BL21 (DE3). The fusion protein GST-SOD1-X-R9 not only has activity of GST and SOD, but also can penetrate cells through R9 in a normal cellular environment and scavenging excessive free radicals in the cells, thereby protecting the cells from oxidative damage.

Description

technical field [0001] The invention relates to the field of fusion proteins, more specifically to the GST-SOD1-X-R9 fusion protein and its preparation method and application. Background technique [0002] Malignant tumors are currently one of the major diseases that seriously affect human health and threaten human life. Cancer, cardiovascular and cerebrovascular diseases and accidents are the three major causes of death in all countries in the world today. Radiation therapy is an effective means of killing tumors, and it undertakes the treatment of 2 / 3 tumor patients. However, the side effects of radiation damage caused by free radicals generated by radiotherapy are large, seriously weakening the body's local and overall resistance, and patients are often forced to interrupt radiotherapy because of the occurrence of these side effects; side effects of radiation damage can also produce some late effects, affecting The quality of life of patients is even life-threatening. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/44A61P39/06A61P39/00A61K47/40
CPCA61K38/00C07K2319/00C07K2319/03C12N9/0089C12N9/1088C12Y205/01018
Inventor 潘剑茹李玲玲
Owner FUZHOU UNIV
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