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A single-chain antibody targeting transferrin receptor in the brain and its application

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

Active Publication Date: 2021-10-29
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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  • A single-chain antibody targeting transferrin receptor in the brain and its application
  • A single-chain antibody targeting transferrin receptor in the brain and its application
  • A single-chain antibody targeting transferrin receptor in the brain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Extraction, amplification and preliminary identification of a single-stranded anti-body weight chain gene and a light chain gene of the brain-targeting transform protein receptor

[0034] The total RNA was extracted: collecting the total heterogeneous tumor cells, and the total RNA was extracted with phenol-chloroform extraction method, dissolved in 20-50 μlDEPC water, and stored -80 ° C.

[0035] RT-PCR synthesis cDNA first chain: Total RNA is a template reverse transcription synthesis cDNA first chain, the kit is purchased in Beijing All-on-Today Biotechnology Co., Ltd., the reaction is carried out according to the product manual.

[0036] PCR amplified light weight chain variable region gene: polymerase is TransStart Fastpfu DNA Polymerase High-fidelity polymerase. The primers used according to the literature (Jian Yi Li, Keijiro Sugimura, Ruben J.Boadoet.al Genetically engineered brain drug delivery vectors:. Cloning, expression and in vivo application of an a...

Embodiment 2

[0044] Example 2 Amplification of a single-chain antibody of the brain-targeted transitional protein receptor according to the present invention

[0045] The heavy-chain gene and the light chain gene are connected to the flexible peptide segment of 15 amino acids (Seq ID NO: 3) by OverLAP PCR.

Embodiment 3

[0046] Example 3 Construction of a single-chain antibody expression vector of the brain targeting of the brake of the iron protein receptor

[0047] The single-chain antibody gene of the iron-converted protein receptor is a template, and the enzyme-containing point primer is formed into PCR amplification. The PCR product was detected by 1.5% agarose gel electrophoresis, and the agarose gel recovery kit was recovered to the target gene. The PCR amplification end product and plasmid PET-28A were digested with restriction endonizers, and the enzyme-digestive product was subjected to a t4 ligase 16 ° C overnight. After connecting, E. coli Transsetta (DE3) Chemically CompeTent Cell induced cells, coating flat plates, subclonal doubles and sequencing identification. Enzyme digestion figure 2 As shown, the positive clone that has been verified by the bacterial liquid PCR has a two strip having a length of about 5000 bp and 700 bp after digestion, indicating that the single-chain antibody...

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Abstract

The invention belongs to the field of genetic engineering antibody technology and cell biology technology, and specifically relates to a single-chain antibody targeting transferrin receptor (TfR) in the brain. In vitro experiments have proved that the single-chain antibody targeting transferrin receptors of the present invention can specifically bind to transferrin receptors, and can be used as a small molecule carrier for diagnosis and treatment of central nervous system diseases, and can be applied to coupling brain It is of great value to the development and research of drugs for central nervous system diseases.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering and cell biology, and more particularly to a brain-targeted single-stranded antibody that can be targeted with human endothelial cells high-specific binding, with specificity to transport drugs to brains. The role is a genetic engineering antibody that can be used in human nervous system disease treatment. Background technique [0002] The central nervous system (CNS) disorder disease has affected more than 1 billion people worldwide, which has exceeded cardiovascular and cerebrovascular diseases and cancer impact on humans. The burden of treatment has taken 13% of global public health burdens. . Because the presence of the blood brain barrier (BBB) ​​makes the drug unable to enter the brain, this is the most important reason for affecting the treatment of central nervous system. The blood-brain barrier consists of vascular endothelial cells, week cells, astrocytes, neuronal vascular units (N...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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