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Application of transthyretin in aspects of entering eye and preparing drop

A technology of thyroxine and drops, applied in the field of medicine, can solve the problems of low delivery efficiency and short half-life of functional small peptides

Active Publication Date: 2020-07-24
易舟(上海)生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The above studies all have certain limitations. Intraocular injection will cause certain trauma, and the delivery efficiency of small functional peptides is low and the half-life is short. Harmful or less harmful drugs and methods of administration

Method used

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  • Application of transthyretin in aspects of entering eye and preparing drop
  • Application of transthyretin in aspects of entering eye and preparing drop
  • Application of transthyretin in aspects of entering eye and preparing drop

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0166] Embodiment 1: the preparation of transthyretin

[0167] Including the following steps:

[0168] (1) Construction of recombinant plasmid pET 21a (+)-His-tag-TTR: His-tag-TTR with synthetic nucleotide sequence as shown in SEQ ID NO: 4 (wherein the amino acid sequence of TTR used is as SEQ ID NO: 1, pET 21a was purchased from ATCC), which was connected to pET 21a (+) with Nde I and EcoR I enzymes, and was successfully constructed after sequencing verification.

[0169] (2) Expression and purification of recombinant TTR: The pET 21a(+)-His-tag-TTR plasmid constructed in step (1) was transformed into E.coli BL21(DE3) cells, and the obtained recombinant E.coli BL21(DE3) Cultivate in LB medium, prepare seed liquid, and then insert 5% inoculum into 5L TB medium, temperature 37°C, stirring paddle speed 150rpm, cultivate to OD 600 1.5-2.0; add 0.1-0.5mM IPTG to induce 8-16h (Table 1). The TTR was prepared by high-pressure homogenization and the supernatant was subjected to aff...

Embodiment 2

[0174] The fusion protein of transthyretin-green fluorescent protein (TTR-GFP) was prepared according to the following method:

[0175] (1) Recombinant plasmid pET 21a(+)-His-tag-TTR-GFP (the following recombinant plasmid pET 21a(+)-His-tag-TTR-X plasmid picture is shown in Figure 4 (shown) construction: Synthesize the His-tag-TTR-GFP sequence shown in SEQ ID NO: 5, connect it to pET 21a (+) with Nde I and EcoR I enzymes, and verify through sequencing that the construction is successful.

[0176] (2) Expression of TTR-GFP fusion protein: Transform the recombinant plasmid constructed in step (1) into E.coli BL21(DE3) cells with a 5% inoculum size, insert 5L TB medium, temperature 37°C, stirrer Rotational speed 150rpm, culture OD 600nm to 1.5-2.0; add 0.1-0.5mM IPTG to induce for 8-16h; use high pressure homogeneously break the bacteria and prepare the TTR-GFP fusion protein by nickel column affinity adsorption on the supernatant. The resulting protein was passed through an e...

Embodiment 3

[0180] The fusion protein (TTR-Lysozyme) of transthyretin-egg white lysozyme was prepared according to the following method:

[0181] (1) Construction of recombinant plasmid pET 21a (+)-His-tag-TTR-Lysozyme: synthesize the His-tag-TTR-Lysozyme sequence shown in SEQ ID NO: 6, and combine it with Nde I and EcoR I enzymes The connection of pET 21a (+) was verified by sequencing, and the construction was successful.

[0182] (2) Expression of TTR-Lysozyme fusion protein: Transform the pET 21a(+)-His-tag-TTR-Lysozyme constructed in step (1) into E.coli BL21(DE3) cells with a 5% inoculum amount, and insert 5L TB medium, temperature 37°C, stirring paddle speed 150rpm, culture OD 600nm to 1.5-2.0; add 0.1-0.5mM IPTG to induce for 8-16h; use high-pressure homogenization to break the bacteria and prepare the TTR-Lysozyme fusion protein through nickel column affinity adsorption on the supernatant. The resulting protein was passed through an endotoxin adsorption column (Pierce TM High ...

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Abstract

The invention provides application of transthyretin in the aspect of serving as a carrier for a protein and / or polypeptide drug to enter an eye through an eye barrier. The transthyretin is a protein consisting of amino acid as shown in SEQ ID NO:1 or a mutation thereof or a modification thereof. The invention further provides application of the transthyretin and / or a fusion protein of the transthyretin and a drug to preparation of a drop and a drop. The drug is the protein and / or polypeptide drug. The transthyretin has good biocompatibility and safety in human bodies, can effectively convey aforeign protein and / or polypeptide into the eye and achieves an effect of treating eye diseases. In an eye dropping manner, when the eye diseases such as DR (Diabetic Retinopathy), AMD (Age-related macular degeneration), ROP (Retinopathy of Prematurity) and the like are treated, the transthyretin can enter a vitreous body and eye ground across a cornea barrier so as to obviously inhibit eyeball retina leakage, obviously reduce the number of retinal new blood vessels and effectively relieve the pathological phenomena of the eye diseases.

Description

technical field [0001] The present invention relates to the field of medical technology, in particular to the application of transthyretin as a carrier for protein and / or polypeptide drugs to enter the eye through the eye barrier, and the present invention also relates to transthyretin and / or transthyretin The application of the fusion protein composed of protein and protein and / or polypeptide medicine in the preparation of drops and a drop. Background technique [0002] Diabetic Retinopathy (DR), referred to as "sugar retinopathy", is the clinical manifestation of diabetic microangiopathy and one of the most serious complications of diabetes. Now it has become one of the main blinding eye diseases. Mainly in the microenvironment of long-term high glucose and hypoxia in the eye, clinical symptoms such as microvascular obstruction, microangioma, hemorrhage, venous dilatation, macular edema, neovascularization, massive vitreous hemorrhage, intraocular fibrosis, and retinal det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/64A61K45/00A61K9/00A61P27/02C07K19/00
CPCA61K9/0048A61K45/00A61K47/64A61K9/0019A61P27/02C07K14/47C07K2319/00Y02A50/30
Inventor 辛瑜
Owner 易舟(上海)生物医药有限公司
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