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Recombinant TAT-XIAP fusion protein

A fusion protein and protein transduction technology, applied in the field of fusion proteins, can solve the problems of viral vector safety, cumbersome operation, and insufficient expression level of target genes

Inactive Publication Date: 2012-04-25
GUILIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional gene therapy is to introduce the target gene into cells and make it express, but this technology has the following shortcomings: First, the conventional transgenic method is cumbersome to operate, the transfection efficiency is low, and the expression level of the target gene after transfection is insufficient , the regulation of gene expression is difficult, followed by the safety problems of commonly used viral vectors
However, XIAP is a macromolecular substance with a molecular weight of 57kDa, and further research and clinical application are greatly limited because it is difficult to pass through the blood-brain barrier.

Method used

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  • Recombinant TAT-XIAP fusion protein
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  • Recombinant TAT-XIAP fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1TAT-XIAP fusion protein

[0036] 1. Construction of pTAT-XIAP fusion protein prokaryotic expression vector

[0037] According to the DNA sequence of rat XIAP provided by the NCBI gene bank, it was optimized according to the preferred codons of Escherichia coli, and the gene sequence was synthesized by Shanghai Sangon Biotechnology Service Co., Ltd., with NcoI and XhoI restriction sites at both ends. The pTAT-HA plasmid was double digested with NcoI and XhoI enzymes, and the XIAP gene was inserted between the NcoI and XhoI restriction sites to construct the pTAT-XIAP fusion protein prokaryotic expression vector. The plasmid vector schematic diagram is as follows figure 1 As indicated, the pET-32a-XIAP expression vector was additionally constructed to produce XIAP as a control.

[0038] 2. Transformation of pTAT-XIAP recombinant plasmid in Escherichia coli

[0039] Take 100 μl of DH5α competent cells in an ice water bath, add 10 μl of TAT-XI...

Embodiment 2

[0048] Example 2 Detection of TAT-XIAP fusion protein's ability to penetrate nerve cell membranes

[0049] Take newborn SD rats, take brains under aseptic conditions, digest with 0.125% trypsin (37°C, 30min) and disperse, then dilute to 5×10 with DMEM medium 5 The cell liquid of 1 cell / ml density is inoculated in the petri dish that is coated with polylysine, adds the TAT-XIAP fusion protein (test group) after the purification of 1~2 μ l embodiment 1 in culture liquid, mixes gently, After culturing for 6-12 hours, make cell slides, detect XIAP by immunocytochemistry (SP method), and observe the ability of the fusion protein to pass through the cell membrane.

[0050] The results showed that as the concentration of the fusion protein increased, the positive staining increased, but no obvious positive staining was seen in the control group treated with XIAP or PBS, indicating that the fusion protein containing the TAT protein transduction domain had entered the brain cells.

Embodiment 3

[0051] Example 3 Detection of the ability of TAT-XIAP fusion protein to cross the blood-brain barrier

[0052] SD rats were injected into the tail vein of 10-20 μl of the purified TAT-XIAP fusion protein (test group), XIAP (negative control group) and PBS (blank control group) in Example 1 respectively for 6-12 hours, and immunohistochemistry (SP method) was performed. ) to detect XIAP in brain tissue slices, and to observe the ability of the fusion protein to pass through the blood-brain barrier.

[0053] The results showed that the positive staining in the test group increased with the increase of the concentration of TAT-XIAP fusion protein, but no obvious positive staining was seen in the control group. It shows that the fusion protein containing the TAT domain enters the brain cells through the blood-brain barrier.

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a fusion protein formed by fusing a protein transduction domain (PTD) coded by TAT (trans-activator of transcription) genes of HIV (human immunodeficiency viruses) and an X-linked inhibitor of apoptosis protein (XIAP) nucleotide sequence. The amino acid sequence of the fusion protein is expressed as SEQ ID No: 1. Compared with the prior art, the original XIAP genes with nucleotide sequence expressed as SEQ ID No: 3 are optimized by using escherichia coli preferred codons so that the original XIAP genes are efficiently expressed in escherichia coli, the defect that the XIAP cannot stride the blood brain barrier or enter the brain to play a role in apoptosis resistance is made up by using the transduction capability of the PTD coded by the TAT genes of the HIV so that the final expression product of the genes can enter the brain tissue cells to play a role in cell apoptosis resistance, and the fusion protein is used for therapy and research of central nervous system diseases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a fusion protein formed by fusion of a protein transduction domain and an optimized X-linked apoptosis inhibitory protein. Background technique [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 this technology has the following shortcomings: First, the conventional transgenic method is cumbersome to operate, the transfection efficiency is low, and the expression level of the target gene after transfection is insufficient , the regulation of gene expression is more difficult, and the second is the problem of safety in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/11C12N15/49
Inventor 夏春波蒋常文秦小云周思刘源劼刘静张晓红林静范才文刘漫君
Owner GUILIN MEDICAL UNIVERSITY
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