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Human endothelial cell cadherins fusion protein, and preparation method and application thereof

A human endothelial cell and fusion protein technology, applied in the field of human endothelial cell-cadherin fusion protein and its preparation, can solve the problems of easy immune response, poor biocompatibility, poor mechanical properties, etc., and achieve simple and easy modification Easy to operate, easy to operate, stable process effect

Inactive Publication Date: 2015-07-29
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, natural biomacromolecules are widely used in the field of tissue engineering due to their good biocompatibility and biodegradability. However, these macromolecules have the disadvantages of poor mechanical properties and instability during use.
Although chemically synthesized polymers have strong mechanical properties, their biocompatibility is poor and they can easily cause immune responses

Method used

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  • Human endothelial cell cadherins fusion protein, and preparation method and application thereof
  • Human endothelial cell cadherins fusion protein, and preparation method and application thereof
  • Human endothelial cell cadherins fusion protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Construction of pcDNA3.1 / VE-cad-Fc eukaryotic expression vector

[0049] According to the human VE-cadherin protein sequence and functional partitions recorded in UniProt, the upstream primer (P1) was designed; 5′-CCGGATATCATGCAGAGGCTCATGATGCTCC-3′, the EcoR Ⅴ restriction site was introduced, and the downstream primer: (P2) 5′-AAGCGGCCGCTCTGGGCGGCCATATC-3′ , the introduction of Not I restriction site. Using synthetic primers to specifically amplify the hVE-cadherin extracellular domain gene, and sequence the DNA, see figure 1 a. 1% agarose gel electrophoresis to analyze the PCR product, the PCR product recovered from the gel was digested by EcoR V and Not I, and inserted between the pcDNA3.1-Fc EcoR V and Not I sites, namely pcDNA3.1 / VE -cadherin-Fc, plasmid pcDNA3.1-Fc was kindly provided by Professor Akaike Toshihiro, Tokyo Institute of Technology, see figure 1 b. Gene sequencing was detected by Nanjing GenScript. The full-length hVE-cad-Fc gene sequence is the se...

Embodiment 2

[0051] Expression and purification of hVE-cad-Fc fusion protein

[0052] The plasmid pcDNA3.1-VE-cadherin-Fc containing the target gene constructed in Example 1 was transfected into 293-6E cells by PEI and kept at 37°C in 5% CO 2 After suspension culture, the cell supernatant was collected after the sixth day, and the hVE-cad-Fc fusion protein was purified by rProtein A FF column. SDS-PAGE electrophoresis analysis showed that the molecular weight of the obtained product was consistent with the predicted theoretical value, see figure 2 a. It has been determined that 10 mg of the target protein can be obtained from 1 L of 293-6E cell culture medium with a purity of about 87%. The resulting fusion protein hVE-cad-Fc is bound to the surface of the hydrophobic material through the Fc end to form an endothelial cell cadherin fusion protein modified for endothelial cell culture, see figure 2 b.

Embodiment 3

[0054] Surface Modification of Hydrophobic Materials by hVE-cad-Fc Fusion Protein, i.e. Substrate Conditions and Its Evaluation

[0055] The maximum immobilization amount and long-term stability of the fusion protein were detected by Elisa method. Dissolve hVE-cad-Fc in PBS to prepare solutions of different concentrations, spread them on polystyrene 96-well plates, incubate at 37°C for 2 hours, wash with PBS for 3 times, then block with 1% BSA for 1 hour at room temperature, and add anti-hVE-cadherin antibody Incubate at 37°C for 1 hour, wash with PBS for 3 times, add horseradish peroxidase-labeled secondary antibody and incubate at 37° for half an hour, wash with PBS for 3 times, add TBA chromogenic solution, incubate at 37°C for 10 minutes in the dark, add stop solution within 10 minutes Microplate reader detection 450nm, see image 3 a. In the hVE-cad-Fc fusion protein stability experiment, the fusion protein incubation time is 1d, 3d, 5d, 7d, the concentration is 10ug / ml...

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Abstract

The invention aims to build an expression plasmid of a fusion protein hVE-cad-Fc of an extracellular structural domain segment of human blood vessel endothelium cadherins protein and an Fc segment of immunoglobulin G by utilizing a genetic engineering technology for the first time, and realize biosynthesis of hVE-cad-Fc fusion protein using eukaryotic cells. The fusion protein forms a stable hVE-cadhrin functional protein layer matrix on the material surface via Fc mediation, so that not only is the hydrophilia of hydrophobic materials improved, but also the property of the endothelial cell selective adhesion to the material surface is greatly improved; in addition, the expression of the endothelial cell proliferation and differentiation functions is improved. According to the invention, the isologous association of the hVE-cad-Fc fusion protein matrix and VE-cadherin positively expressed on the cell surface activates the intercellular adhesion link signal path, and through the synergistic effect with the blood vessel endothelium cell growth factors, the expression of the adhesion, proliferation, anti-apoptosis and differentiation functions of the blood vessel endothelium cells are regulated and controlled; the hVE-cad-Fc fusion protein provided by the invention can be used to improve the affinity of the material endothelium cells, promote the functional modification of artificial extracellular matrix subjected to tissue engineering vascularization / endothelialization and biomedical materials.

Description

technical field [0001] The invention relates to the technical field of research and development of biological materials and their functionalization, in particular to a human endothelial cell cadherin fusion protein and its preparation method and application. Background technique [0002] In recent years, the research and application of bioactive materials in the field of tissue engineering has attracted widespread attention, especially with the development of tissue engineering and regenerative medicine, biomaterials based on protein engineering have gradually become an important tool for special biological and medical research. new hotspots. Anticoagulation and vascularization of materials have always been one of the bottlenecks in tissue engineering and regenerative medicine research, especially for regenerative tissues with a size of more than 200 μm, insufficient vascularization will lead to insufficient supply of oxygen and nutrients to cells lead to apoptosis inside t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85C12N5/071A61L27/34A61L27/50
Inventor 杨军许可高超
Owner NANKAI UNIV
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