Application of transthyretin in transferring fusion protein into eyes

A technology of thyroxine protein and protein, which is applied in the application field of transthyretin carrying exogenous protein to the eye through the eye barrier, which can solve the problems of short half-life and low delivery efficiency, and achieve good biocompatibility and safety effect

Inactive Publication Date: 2020-04-07
TONGYAN (SHANGHAI) MEDICAL EQUIPMENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] All the above studies have certain limitations. Intraocular injection will cause certain trauma, and the delivery efficiency of functional small peptid

Method used

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  • Application of transthyretin in transferring fusion protein into eyes
  • Application of transthyretin in transferring fusion protein into eyes
  • Application of transthyretin in transferring fusion protein into eyes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of transthyretin: comprising the following steps:

[0031] (1) Construction of recombinant plasmid pET 21a(+)-TTR: Synthesize the nucleotide sequence shown in SEQ ID NO.2, connect it to pET 21a(+) with Nde I and EcoR I enzymes, and verify by sequencing , build succeeds.

[0032] (2) Expression and purification of recombinant TTR: transform the pET 21a(+)-TTR plasmid constructed in step (1) into E.coliBL21(DE3) cells, and culture recombinant E.coli BL21(DE3) in LB medium , prepare the seed solution, and then insert it into 5L TB medium with a 5% inoculum amount, at a temperature of 37°C, with a stirring paddle speed of 150rpm, and 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 affinity adsorption on a nickel column. The resulting protein was passed through an endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal SpinColum...

Embodiment 2

[0036] The fusion protein of transthyretin-green fluorescent protein (TTR-GFP) was prepared according to the following method:

[0037] (1) Construction of recombinant plasmid pET 21a(+)-TTR-GFP: Synthesize the TTR-GFP sequence shown in SEQ ID NO.3, connect it to pET 21a(+) with Nde I and EcoR I enzymes, and pass Sequencing verification, the construction was successful.

[0038](2) Expression of TTR and GFP fusion protein: Transform the recombinant plasmid constructed in step (1) into E.coli BL21 (DE3) cells of pET 21a (+)-TTR-GFP plasmid with a 5% inoculation amount, 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 homogeneously break the bacteria and prepare the TTR-GFP fusion protein by nickel column affinity adsorption on the supernatant (Table 2). The resulting protein was passed through an endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Sp...

Embodiment 3

[0043] A method for fusion expressing transthyretin to transport exogenous protein into the eye through the eye barrier, the method comprising the following steps:

[0044] (1) Construction of recombinant plasmid pET 21a(+)-TTR-Lysozyme: synthesize the TTR-Lysozyme sequence shown in SEQ ID NO.4, connect it to pET 21a(+) with Nde I and EcoR I enzymes, and Sequencing verification, the construction was successful.

[0045] (2) Transform the pET 21a(+)-TTR-Lysozyme constructed in step (1) into E.coli BL21(DE3) cells with a 5% inoculum amount, insert 5L TB medium at 37°C, and stirrer 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 by nickel column affinity adsorption on the supernatant (Table 3). The resulting protein was passed through an endotoxin adsorption column (Pierce TM HighCapacity Endotoxin Removal Spin Columns, ThermoFisher) to remove ...

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Abstract

The invention discloses application of transthyretin in transferring fusion protein into eyes, belonging to the technical field of protein engineering. The invention provides a method for allowing foreign protein to pass through eye barriers and to be conveyed into eyes through fusion expression of transthyretin as shown in SEQ ID NO.1 and the foreign protein. The results of animal experiments prove that as the fusion protein prepared in the invention is used for eye drop treatment on rats and rabbits, foreign protein can be rapidly and effectively conveyed to fundus. The transthyretin used inthe method is derived from a human body, has good biocompatibility and safety in the human body, and has important application prospects when used as a substitute of injection drugs in the field of medicine.

Description

technical field [0001] The invention relates to the application of transthyretin transport fusion protein into the eye, especially the application of transthyretin carrying exogenous protein to the eye through the eye barrier, and belongs to the technical field of protein engineering. Background technique [0002] The treatment of eye diseases requires the effective delivery of drugs into the eye, especially age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, glaucoma-induced nerve changes, etc.; however, there are many barriers in the eye , the effective delivery of drugs into the eye is full of challenges. Therefore, ocular drug delivery systems have received extensive attention recently. At present, eye drops are the most widely used dosage form in the clinical treatment of eye diseases, but they are quickly lost from the ocular surface, and their bioavailability is relatively low. In response to this situation, the formulations of microspheres...

Claims

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Application Information

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IPC IPC(8): A61K47/64A61K45/00A61K9/00A61P27/02C07K19/00
CPCA61K9/0048A61K45/00A61K47/64A61K9/0019A61P27/02C07K14/47C07K2319/00Y02A50/30
Inventor 张允斌辛瑜
Owner TONGYAN (SHANGHAI) MEDICAL EQUIPMENT CO LTD
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