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A kind of tat PTD-Endostatin-RGD recombinant protein and its preparation method and application

A recombinant protein and sequence technology, which is applied to peptide/protein components, chemical instruments and methods, recombinant DNA technology, etc., can solve the problems of no fusion expression, improve targeting and residence time, save enzyme digestion recovery, Effect of Rapid DNA Ligation Reaction

Active Publication Date: 2019-03-08
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are still a series of technical difficulties in combining target proteins with different activities for fusion expression, such as whether different proteins can still have the original activity after integration, the choice of fusion protein expression system, etc. Therefore, there is currently no A report on the fusion expression of Tat PTD, Endostatin and RGD

Method used

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  • A kind of tat PTD-Endostatin-RGD recombinant protein and its preparation method and application
  • A kind of tat PTD-Endostatin-RGD recombinant protein and its preparation method and application
  • A kind of tat PTD-Endostatin-RGD recombinant protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Preparation of Tat PTD-Endostatin-RGD fusion protein

[0053] 1. The construction of fusion gene: adopt the method for PCR to obtain fusion gene with Tat PTD-Es as template (results see Figure 1a-Figure 1b ), and recycle.

[0054] 2. Double digestion of the empty plasmid pET28a: the E. coli expression plasmid pET28a was double digested with BamH I and Xho I, and the fragments were recovered.

[0055] 3. Ligation of the fusion gene and the plasmid: Add 100 ng of the recovered product from PCR and 100 ng of the product recovered from the digestion of the empty plasmid into the Gibson polymerization ligation reaction system, react at 50°C for 1 h, and the ligated product is used for the next transformation.

[0056] 4. Transformation of recombinant plasmids: quickly add 6 μl of the above ligation solution to 100 μl of pre-cooled competent cells E.coliDH5α and mix well, ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, ice-bath for 2-3 minutes, add...

Embodiment 2

[0065] Embodiment 2: Research on the biological activity of Tat PTD-Endostatin-RGD fusion protein

[0066] Investigate the activity of Tat PTD-Es-RGD in inhibiting the proliferation of endothelial cells: with Es, Tat PTD-Es, Es-RGD as contrast (here as the other three proteins of contrast as the preparation method of TatPTD-Es-RGD is the same), adopt The CCK-8 method was used to investigate the activity of the purified Tat PTD-Es-RGD in inhibiting the proliferation of vascular endothelial cells. The specific implementation steps were as follows:

[0067] The cryopreserved solution of human umbilical vein endothelial cells (EAHY926) was quickly thawed in a 37°C water bath, centrifuged at 800r / min for 3min, and the supernatant was discarded. Resuspend the cell pellet with sterile PBS, centrifuge at 800r / min for 3min, and discard the supernatant. The cell pellet was resuspended with DMEM medium (containing 10% FBS) and blown evenly to form a cell suspension, and the cell count w...

Embodiment 3

[0070] Example 3: Inhibitory effect of Tat PTD-Es-RGD on the generation of chicken embryo chorioallantoic membrane vessels (CAM)

[0071] 1. Test method:

[0072] 1.1 The method of incubating CAM: the method is as follows: select well-growth white preserved eggs from breeder farms with a fertilization rate greater than 90% (clean and smooth surface, uniform size, standard egg shape, uniform stomata and air cells), and use them after cleaning. 1‰ benzalkonol was used to wipe and disinfect the shell of the eggs, and the eggs were cultured in an insulated electric heating constant temperature incubator at a temperature of (38±1)°C and a humidity of 60-80%, with certain ventilation condition. The air cell of the egg is upward, and the long axis is at an angle of about 70°-80° to the egg tray. Turn the egg 3 times a day, and the angle of the egg turning should be 45° in the horizontal position. After the eggs were incubated for 5 days, the eggs were illuminated with an egg lamp,...

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Abstract

The invention discloses Tat PTD-Endostatin-RGD recombinant protein which is composed of the protein transduction domain of transactivation transduction protein Tat of human immunodeficiency viruses, the human endostatin and RGD tripeptide coming from the fiber cell attachment domain. The invention further discloses a preparation method of the Tat PTD-Endostatin-RGD recombinant protein, and application of the Tat PTD-Endostatin-RGD recombinant protein to medicine for treating diseases caused by new vessels and medicine for restraining endothelial cell migration. The Tat PTD-Endostatin-RGD recombinant protein reserves the activity of endostatin for restraining new vessel proliferation, and can penetrate through blood brain barriers or eyeball barriers to be combined with integrin specificity over-expressed at new vessels, the targeting performance of the fused protein is greatly improved, and the retention time of the fused protein is greatly prolonged.

Description

technical field [0001] The invention relates to a Tat PTD-Endostatin-RGD recombinant protein, a preparation method and application thereof, and belongs to the technical field of biopharmaceuticals. Background technique [0002] Endostatin (Endostatin, Es) is an endogenous angiogenesis inhibitor isolated from hemangioendothelioma by O'Reilly et al. in 1997. Endostatin is a fragment of the C-terminus of collagen XVIII molecule, with a total of 184 amino acid residues and a molecular weight of 20kDa. Studies have shown that endostatin can only inhibit the formation of new blood vessels but not the existing blood vessels. Endostatin is currently the most effective tumor angiogenesis inhibitor with the best experimental effect. It has attracted much attention in recent years. At present, phase I and phase II clinical trials have been carried out in the United States. A class of new drug Endostar (Endostar) with national independent intellectual property rights has been develope...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/39A61K47/42A61P35/00A61P9/10
Inventor 王凤山李妍生举正冯丹阳
Owner SHANDONG UNIV
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