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Peptide library penetrating through blood brain barrier and screening method thereof

A technology of blood-brain barrier and peptide library, applied in the direction of peptide library, peptide, library, etc., can solve difficult brain diseases, effective treatment, difficult to reach the target of action, etc., achieve reduced dose, high success rate, improved drug distribution effect

Active Publication Date: 2019-04-23
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The barrier mechanism of the blood-brain barrier, which strictly restricts the entry of substances between the brain and the outside world, will make it difficult for most current effective drugs to reach the target, and it is difficult to effectively treat brain diseases

Method used

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  • Peptide library penetrating through blood brain barrier and screening method thereof
  • Peptide library penetrating through blood brain barrier and screening method thereof
  • Peptide library penetrating through blood brain barrier and screening method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, building a database

[0055] 1. Based on the sequence analysis of the kunitz region of the active center, the following central skeleton sequence was found, and the construction of the phage-displayed polypeptide library kun-M was designed and completed.

[0056] Build method:

[0057] Library 1: TFYGGRXKRNNF XXXXX (SEQ ID NO: 1)

[0058] X is a random amino acid.

[0059] Long Primer:

[0060] Library1

[0061] 5'-cccaggtgcag ctgcag ACCTTTTATGGTGGTCGTNNKAAACGTAATAATTTTTNNKNNKNNKNNKNNK tctaga ggggacccaggtc-3' (SEQ ID NO: 2)

[0062]Said N is A, T, C or G; said K is G or T

[0063] The restriction sites are underlined, and the uppercase letters are the nucleic acid sequences encoding the peptide library.

[0064] Design the first pair of primers NAG-F: GCCCAGGTGCAGCTG (Tm 57.24) (SEQ ID NO: 3)

[0065] The second pair of primers NAG-R: GACCTGGGTCCCCTTAG (Tm 57.29) (SEQ ID NO: 4)

[0066] The primers were synthesized by Primer Synthesis Company. ...

Embodiment 2

[0102] Example 2. In vivo screening of peptide library

[0103] Before the screening, the pre-experiment was conducted to determine the optimal time for enriching phage display peptides in the mouse brain. The results were as follows: figure 2 shown. 24h after inoculation, the brain-to-blood ratio of phage was the highest, indicating the highest enrichment degree in the brain.

[0104] The screening method is as follows:

[0105] 1 Adult balb / c mice (18-22g), take the phage library TBS and dilute it to 100ul / 1011PFU, and inject it into the tail vein. According to the preliminary experiment, the brain-blood ratio of phage enrichment in the brain is the highest at 24 hours.

[0106] 2 Take the time point of 24 hours, anesthetize with 5% chloral hydrate, keep the mice sterile, flow through the heart with 100ml of normal saline, dissect and take the brain, homogenize the brain reticular body, break it up by ultrasonic, centrifuge, pass through a 0.45μm filter membrane, Take th...

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Abstract

The invention relates to a peptide library penetrating through a blood brain barrier, a method for selecting blood brain barrier penetrating peptides by using the peptide library, and obtained blood brain barrier penetrating peptides. The invention also provides a coding nucleic acid, a nucleic acid construct, a host cell of the blood brain barrier penetrating peptides, and the application as a brain drug delivery carrier in preparing brain drugs. Based on the previously known structure-activity relationship and bioinformatics analysis of the blood brain barrier penetrating peptides, a methodfor efficiently screening and preparing the blood brain barrier penetrating peptides is provided by fully utilizing the blood brain barrier function of living animals and phage surface peptide librarydisplaying technology.

Description

technical field [0001] The present invention relates to the fields of bioengineering and medicine, in particular to the fields of phage surface display technology, molecular biology technology, and drug delivery technology, in particular to a peptide library that passes through the blood-brain barrier, and uses the peptide library to screen the blood-brain barrier Methods of peptides, obtained blood-brain barrier-penetrating peptides and uses thereof. Background technique [0002] The brain is the high-level nerve center of humans. There is a barrier between plasma and brain cells formed by brain capillary walls and glial cells called the blood-brain barrier, which limits the entry of various substances into the brain. Experimental and clinical evidence suggests that the blood-brain barrier maintains a stable chemical environment for neurological function and protects the brain from harmful substances. The blood-brain barrier is thus important for many complex problems, suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/10C12N15/11C07K7/08C12N15/70C12N1/21G01N33/68A61K47/62
CPCA61K47/42G01N33/68C12N15/11C12N15/70C40B40/10C07K7/08G01N2410/00
Inventor 林坚李成鹏陈龙李朝刚
Owner PEKING UNIV
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