Fusion protein with protein transduction structure field TAT-PTD and application thereof

A fusion protein and sequence technology, applied in the field of genetic engineering, can solve problems such as reducing application value, and achieve the effects of improving antioxidant capacity, high-efficiency antioxidant drugs, and high application value

Inactive Publication Date: 2007-02-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the human body is subjected to oxidative damage, these antioxidant enzymes supplied by exogenous sources cannot penetrate the cell membrane and enter the cells to scavenge free radicals and protect cells, which greatly reduces the application value of these enzymes

Method used

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  • Fusion protein with protein transduction structure field TAT-PTD and application thereof
  • Fusion protein with protein transduction structure field TAT-PTD and application thereof
  • Fusion protein with protein transduction structure field TAT-PTD and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Expression of HIV-TAT-SOD-1 fusion protein in Escherichia coli

[0038] 1. Construction of recombinant TAT-SOD-1 fusion protein gene

[0039] 1) Amplify the SOD-1 gene with RT-pCR

[0040]The total RNA of the liver was extracted with TRIzol, and PCR amplification was performed with primers P1 and P2 (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.), P1: 5'-CTCGAGGCGACGAAGGCCGTGTGCGTG-3' (SEQ ID NO: 8), and P2 : 5'-GGATCCTTATTGGGCGATCCCAATTAC-3' (eg, SEQ ID NO: 9). The PCR amplification reaction was carried out in a 50 microliter system, and the conditions were denaturation at 94°C for 5 min, 30 s at 94°C, 1 min at 54°C, Imim at 72°C, 30 cycles, and 5 min at 72°C. After the reaction was completed, the entire PCR reaction solution was added to 1% agarose for electrophoresis, and a 500 bp band was recovered by tapping the gel (the recovery reaction used Saibaisheng Company's recovery kit). The recovered product was connected to the T...

Embodiment 2

[0047] Example 2 Detection of TAT-SOD-1 fusion protein antioxidant effect in Hela cells

[0048] In the culture medium of Hela cells cultured in vitro, 0.1-2 μl of purified TAT-SOD-1 protein was added to the test group, and 0.1-2 μl of purified SOD-1 protein was added to the control group, and the medium was replaced two hours later. Methyl viologen (Paraquat) was added at a final concentration of 5 mM to generate superoxide anion. Cell viability was measured 12 hours later by colorimetry. Colorimetric method adopts MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dip-henyltetrazolium bromide, Sigma) to measure. The results show that the cell survival rate of the test group adding TAT-SOD-1 protein is obvious increased, confirming that the TAT-SOD-1 fusion protein has entered the cell and has obvious antioxidant capacity ( Figure 4 shown).

Embodiment 3

[0049] Example 3 Detection of TAT-SOD-1 fusion protein's antioxidant effect in keratinocytes

[0050] In the culture medium of primary cultured mouse skin keratinocytes, add 0.5-2 μl purified TAT-SOD-1 protein to the test group, add 0.5-2 μl purified SOD-1 protein to the control group, and use trypsin after two hours Digest the cells with EDTA mixture, centrifuge to remove the supernatant, add phosphate buffer solution, place on a vortex mixer for vigorous mixing, and centrifuge again, the supernatant is the SOD-1 extract. Adding NaOH to dimethyl sulfoxide can generate oxygen free radicals under aerobic conditions. At this time, adding chemiluminescent agent luminol will emit cold chemiluminescence. Add 0.1ml of SOD-1 extract to the luminescence solution, and calculate the luminescence inhibition rate. The higher the inhibition rate, the higher the concentration of SOD-1. The results showed that the SOD-1 concentration of the test group was significantly higher than that of ...

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Abstract

The invention discloses a fusing protein with SOD-1, HO-1, CAT and PHGPx in the genetic engineering technological domain, which displays excellent resistance to oxidation when the fused protein enters into cells through cell membrane.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a fusion protein formed by a protein transduction domain and a type of anti-oxidation protein, and the application of the fusion protein in the preparation of anti-oxidation drugs. technical background [0002] Gene therapy is a new technology developed since the 1980s. It mainly introduces DNA or RNA fragments of exogenous genes into target cells or tissues to correct or compensate for gene defects, shut down or Inhibit abnormally expressed genes, so as to achieve the purpose of treatment. [0003] Traditional gene therapy is to introduce the target gene into cells and make it express, but there are many shortcomings in this technology that limit its application. The first is that the transfection efficiency is low, or the expression level of the target gene is insufficient after transfection, and the second is that the commonly used viral vectors have sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/16A61P39/06
Inventor 卢大儒张舒羽
Owner FUDAN UNIV
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