A kind of specific dectin-1 nanobody and its preparation method and application

A dectin-1, nanobody technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of complex production process of antifungal drugs, low bioavailability, poor anti-inflammatory effect, etc. To achieve the effect of poor anti-inflammatory effect, high bioavailability and fast onset

Active Publication Date: 2022-05-24
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art, design and propose a specific Dectin-1 nanobody and its preparation method and application, to solve the problem of low bioavailability, high toxicity, poor water solubility, and stability of existing antifungal drugs. The problems of poor sex, low activity, poor affinity, slow onset, and poor anti-inflammatory effect can overcome the defects of complex production process, high cost, difficult storage, and low yield of existing antifungal drugs, and solve the bioavailability of fungal keratitis drugs Low, poor penetration, high corneal toxicity

Method used

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  • A kind of specific dectin-1 nanobody and its preparation method and application
  • A kind of specific dectin-1 nanobody and its preparation method and application
  • A kind of specific dectin-1 nanobody and its preparation method and application

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Embodiment 1

[0025] The preparation method of the specific Dectin-1 Nanobody involved in this example includes 3 process steps:

[0026] (1) Recovery of nanobody phage library, panning and identification of specific nanobody phage: Infect the nanobody phage library that has been successfully constructed with helper phage M13K07 to obtain phagemids released from the host bacteria, followed by After three rounds of Dectin-1 extracellular antigen panning at concentrations of 100ug / mL, 10ug / mL, and 1ug / mL, polyclonal phage indirect ELISA was performed to verify that the specific Nanobody phages were enriched; the third round of panning was performed. The obtained specific Nanobody phage was spread into a petri dish, 95 single colonies were picked, and the phage carrying the monoclonal gene of the Nanobody specific for Dectin-1 was panned out, and the phage was detected by the indirect ELISA method of monoclonal phage. The specificity was verified to determine the phage with the strongest speci...

Embodiment 2

[0033] The application of the specific Dectin-1 nanobody involved in this example, the specific Dectin-1 nanobody is applied to ophthalmology for the treatment of fungal keratitis, which has the advantages of high bioavailability, good penetration and good specificity , Corneal toxicity is small, anti-inflammatory effect is good. For the preparation of specific Dectin-1 nanobody eye drops, the composition and its content are as follows: the content of specific Dectin-1 nanobody contained in each 1 mL buffer solution is 1 mg.

[0034] The anti-inflammatory effect of the eye drops prepared in this example was verified: after anesthetized mice by intraperitoneal injection of 8% chloral hydrate (0.4 mL / kg), the central corneal epithelial tissue of the right eye with a diameter of about 2 mm was scraped with a lamellar knife to form For corneal defects, 10 μL of Aspergillus fumigatus hyphae stimulated droplets were placed on the cornea, covered with a homemade soft contact lens, an...

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Abstract

The invention belongs to the technical field of biology and immunology, and relates to a specific Dectin-1 nanobody and its preparation method and application. The preparation method of the specific Dectin-1 nanobody comprises three process steps: the preparation of the nanobody phage text library Recovery, panning and identification of specific nanobody phage, construction of recombinant pET‑28a plasmid expression vector, induction of expression and purification of specific Dectin‑1 nanobody; prepared specific Dectin‑1 nanobody against Dectin‑1 recombination Protein, HCECs and RAW264.7 cells are specific; Dectin‑1 nanobody has simple production process, low cost, high yield, easy storage, and simple structure; Dectin‑1 nanobody is applied to the treatment of fungal keratitis and is bioavailable High penetration, good specificity, low corneal toxicity, good anti-inflammatory effect.

Description

Technical field: [0001] The invention belongs to the technical fields of biology and immunology, and relates to a specific Dectin-1 nanobody and a preparation method and application thereof. A novel specific Dectin-1 nanobody and its coding sequence, a vector and host cell for expressing the antibody, the steps and optimization of expressing the nanobody, and using the prepared specific Dectin-1 nanobody for fungal cornea The anti-inflammatory effect in fungal keratitis was verified. Background technique: [0002] Fungal keratitis (FK) is a serious blinding eye disease caused by pathogenic fungal infection. Plant trauma provides opportunities for the occurrence of fungal keratitis, resulting in a high incidence of fungal keratitis. No less. The current clinically used antifungal drugs have shortcomings such as low bioavailability, poor penetration, and high corneal toxicity. However, surgical treatment also has the problems of lack of corneal donors, large postoperative re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/70A61K39/395A61K9/08A61P27/02A61P31/02A61P31/10A61P29/00
CPCC07K16/2851C07K16/005C12N15/70A61K9/08A61K9/0048A61P27/02A61P31/02A61P31/10A61P29/00C07K2317/569C07K2317/565A61K2039/505
Inventor 林静赵桂秋彭旭东李翠刘星王谦
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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