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Tat PTD-Endostatin recombination protein, preparation method and application thereof

A recombinant protein and transduction protein technology, applied in the field of protein transduction, can solve the problems of increasing the economic burden of patients, large dosage, poor cell penetration ability, etc. over effect

Active Publication Date: 2014-07-30
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ES still has some disadvantages. Due to its short half-life in vivo and poor ability to enter cells, the clinical dosage is relatively large, which increases the economic burden of patients.

Method used

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  • Tat PTD-Endostatin recombination protein, preparation method and application thereof
  • Tat PTD-Endostatin recombination protein, preparation method and application thereof
  • Tat PTD-Endostatin recombination protein, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 adopts yeast expression system to express Tat PTD-ES fusion protein

[0055] a. Amplification of Tat PTD-ES fusion gene: 33 codons encoding 11 amino acids of Tat-PTD and a selected enzyme cutting site (EcoR Ⅰ) were designed to the 5′ end of the upstream primer, and PCR technology was used to extract the endostatin-containing pGAPZ α The Tat PTD-ES fusion gene is amplified in a plasmid (owned by the inventor's laboratory and constructed by conventional methods), and the fusion gene fragments obtained by the amplification are about 600, and the PCR results are shown in figure 1 .

[0056] b. Construction of an expression plasmid containing the Tat PTD-ES fusion gene:

[0057] ①pGAPZ α A. Amplification and extraction of yeast expression plasmids: DH5α was routinely activated to prepare competent cells, and pGAPZ was taken α A. Plasmid DNA heat shock method was used to transform DH5α. After transformation, the bacterial solution was spread on a low-salt LB ...

Embodiment 2

[0065] Embodiment 2 adopts Escherichia coli to express Tat PTD-ES fusion protein

[0066] a. Double digestion of fusion gene and empty plasmid: Nde Ⅰ and BamH Ⅰ were used to perform double digestion of Tat PTD-ES fusion gene (constructed by conventional methods) and pET28a expressed in the large intestine, and the fragments were recovered respectively.

[0067] b. Ligation of fusion gene and plasmid: Mix the recovered target gene and plasmid in the ligation system, incubate overnight at 4°C, and the ligation product is used for the next step of transformation.

[0068] c. Transformation of recombinant plasmids: Add 200 μl of E.coli JM109 competent cells to the above ligation reaction solution, mix well, ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, ice-bath for 2 minutes, add 0.8ml LB medium, incubate at 37°C for 1 hour, Spread the incubation solution on the LK plate and incubate at 37°C for 12-16h.

[0069] d. Identification of positive recombinants: Randomly s...

Embodiment 3

[0074] Example 3 The technical process of the renaturation of the inclusion body protein expressed by Escherichia coli

[0075] a. Inclusion body solubilization: after cell wall breaking and ultrasonication, centrifuge at 10,000g for 30 minutes, discard the supernatant to obtain inclusion bodies; wash with urea or guanidine hydrochloride solution containing detergent for several times to remove adherent impurities; then use denaturation The solution was used to dissolve the inclusion bodies, stirred at room temperature for 2 hours, centrifuged at 10000 g for 30 minutes, and the supernatant was the inclusion body solution. The detergent solution is urea with a concentration of 2mol / L, and the detergent solution also contains Triton x-100 at a concentration of 0.5% (v / v). Denaturation solution contains 20-100mmol / LTris / HCl, 8mol / L urea, 5mmol / LEDTA and 10mmol / LDTT. SDS-PAGE results such as Figure 7 shown.

[0076] b. Protein renaturation: After the renaturation buffer is fil...

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Abstract

Disclosed is a Tat PTD-Endostatin recombinant protein, which is a fusion protein comprising a protein transduction domain of human immunodeficiency virus transactivation transduction protein Tat and human endostatin. The amino acid sequence of the protein transduction domain of the human immunodeficiency virus transactivation transduction protein Tat is represented by SEQ ID NO.1, and the amino acid sequence of the human endostatin is represented by SEQ ID NO.2. The protein of the present invention maintains the function that the endostatin can inhibit angiogenesis, has advantages of high transduction efficiency, easy blood-brain barrier crossing, and easy blood-ocular barrier crossing, overcomes the limitation of the poor membrane spanning effect of the endostatin, can play the angiogenesis inhibition effect better, and can be used for treatment of various diseases caused by angiogenesis, comprising ocular vascular proliferative diseases and various tumors, such as diabetes induced retinopathy, non-small cell lung cancer, and the like.

Description

technical field [0001] The invention provides a Tat PTD-Endostatin recombinant protein and its preparation method and application, belonging to the field of protein transduction. Background technique [0002] Endostatin (Endostatin, ES) is a potent inhibitor of endogenous vascular endothelial cell proliferation first discovered by O'Reilly et al. in 1997. Studies have found that ES can specifically inhibit the formation of neovascular endothelial cells, and has inhibitory effects on the generation of neovascular endothelial cells of various origins, and does not affect resting vascular endothelial cells, has no drug resistance, and has little toxic side effects. my country has developed endostatin derivatives into a national first-class new drug Endostar (Endostar, YH16) with independent intellectual property rights for the treatment of non-small cell lung cancer. In addition, researchers have also made some gratifying achievements in the prevention and treatment of ocular ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N15/70A61K38/17A61K47/48A61P35/00A61P9/00A61P27/02
CPCA61K38/00C07K2319/10A61K47/48315C07K14/78A61K47/645A61P9/00A61P27/02A61P35/00
Inventor 王凤山张新科程艳娜谭海宁李妍
Owner SHANDONG UNIV
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