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Single-chain antibody and application of brain targeted transferrin receptor

A technology of transferrin and single-chain antibody, applied in the field of genetic engineering and cell biology

Active Publication Date: 2018-11-23
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, no brain-targeted antibody has been approved by the FDA for marketing, so brain-targeted TfR antibodies have great development potential to become functional antibodies for the treatment of brain diseases

Method used

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  • Single-chain antibody and application of brain targeted transferrin receptor
  • Single-chain antibody and application of brain targeted transferrin receptor
  • Single-chain antibody and application of brain targeted transferrin receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Extraction, amplification and preliminary identification of the heavy chain gene and light chain gene of the brain-targeted transferrin receptor single-chain antibody of the present invention

[0034] Extraction of total RNA: Collect hybridoma cells in logarithmic phase, extract total RNA by phenol-chloroform extraction method, dissolve in 20-50 μl DEPC water, and store at -80°C.

[0035] RT-PCR synthesis of the first strand of cDNA: The first strand of cDNA was synthesized by reverse transcription using total RNA as a template. The kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the reaction was carried out according to the product manual.

[0036] PCR amplification of light and heavy chain variable region genes: the polymerase is TransStart FastPfu DNA Polymerase high-fidelity polymerase. The primers used were based on the literature (Jian Yi Li, Keijiro Sugimura, Ruben J.Boadoet.al. Genetically engineered brain drug delivery vectors: ...

Embodiment 2

[0044] Example 2 Amplification of the single-chain antibody targeting transferrin receptor in the brain according to the present invention

[0045] The heavy chain gene and the light chain gene were connected together through a flexible peptide segment consisting of 15 amino acids (see SEQ ID NO: 3 for its sequence) by Overlap PCR.

Embodiment 3

[0046] Example 3 Construction of the brain-targeted transferrin receptor single-chain antibody expression vector of the present invention

[0047] Using the single-chain antibody gene targeting the transferrin receptor in the brain as a template, design primers containing restriction sites for PCR amplification. The PCR product was detected by 1.5% agarose gel electrophoresis, and the target gene was recovered by the agarose gel recovery kit. The final product of PCR amplification and the plasmid pet-28a were double-digested with restriction endonucleases, and the digested products were gel-recovered and then ligated with T4 ligase overnight at 16°C. After ligation, transform into Escherichia coli Transetta (DE3) Chemically Competent cell competent cells, smear the plate, and select single clones for double enzyme digestion and sequencing identification the next day. Digestion results such as figure 2 As shown, the positive clones that have been preliminarily verified by ba...

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Abstract

The invention belongs to the technical field of genetic engineering antibody and cell biology, and particularly relates to a single-chain antibody of a brain targeted transferrin receptor (TfR). In-vitro experiments demonstrate that the single-chain antibody of the transferrin targeted receptor provided by the invention can be specifically bound to the transferrin receptor, can be used as a smallmolecule carrier for diagnosing and treating central nervous system diseases, and is applied to the conjugation of brain therapeutic drugs so as to transport drugs or diagnostic agents to the brain through transferring endocytosis effects, with a great value for the development and research of drugs for the central nervous system diseases.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and cell biology, and in particular relates to a brain-targeting single-chain antibody capable of highly specifically binding to transferrin receptors on human brain endothelial cells, capable of specifically transporting drugs to the brain It is a genetically engineered antibody that can be used in the treatment of human nervous system diseases. technical background [0002] Central nervous system (CNS) disorders have affected the health of more than 1 billion people worldwide, surpassing the impact of cardiovascular and cerebrovascular diseases and cancer on humans, and their treatment burden has accounted for 13% of the global public health burden . Due to the existence of the blood-brain barrier (BBB), drugs cannot enter the brain to play a role, and this is the main reason that affects the treatment effect of central nervous system diseases. The blood-brain barrier is composed ...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61P25/28
CPCC07K16/28C07K2317/33C07K2317/56C07K2317/565C07K2317/622C07K2317/92
Inventor 刘煜张楚麟李晓蕊杨玥
Owner CHINA PHARM UNIV
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