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Method for constructing expression library of cell penetrating peptide by utilizing bacteriophage display technology

A phage display and penetrating peptide technology, applied in the field of biomedicine, can solve the problems of low positive rate, labor and time consumption, etc.

Inactive Publication Date: 2018-11-06
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, our previous work has confirmed that for the screening and verification of random prokaryotic expression libraries, there are outstanding problems that the positive rate is very low, and it consumes a lot of manpower and time.

Method used

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  • Method for constructing expression library of cell penetrating peptide by utilizing bacteriophage display technology
  • Method for constructing expression library of cell penetrating peptide by utilizing bacteriophage display technology
  • Method for constructing expression library of cell penetrating peptide by utilizing bacteriophage display technology

Examples

Experimental program
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Effect test

Embodiment 1

[0068] A method for constructing a cell-penetrating peptide expression library using phage display technology, using phage display technology, combined with the whole-cell screening mode, to screen polypeptides with cell membrane penetrating activity; and then using the screened cell-penetrating peptides to construct cell-penetrating peptides Prokaryotic expression library.

[0069] The principle of the phage display technology described in the present invention is to recombine the DNA fragment encoding the polypeptide with the gene encoding the phage capsid protein, and express it on the surface of the phage in the form of fusion protein. Based on the affinity between biomolecules and target molecules, through the repeated process of adsorption-elution-amplification, phages containing specific binding to target molecules are screened from phage libraries expressing various foreign proteins, and then enriched, Amplification and gene sequence determination to obtain the amino a...

Embodiment 2

[0108] Example 2: Screening of cell-penetrating peptides by phage display technology

[0109] Titer determination of the first step phage display cyclic heptapeptide library

[0110] Inoculate a single colony of E.coli (Escherichia coli) ER2738 in 5ml LB medium, shake vigorously (225rpm) at 37°C, and cultivate to mid-logarithmic phase (OD 600 value around 0.5). Thaw the top agar in a microwave oven and store at 45°C until use. Divide into 3-4ml aliquots before use, pour into sterilized glass test tubes, one tube for each phage dilution. Take 1 μl of phage random cyclic heptapeptide library, and make 10-fold serial dilution in LB, the dilution range is 10 9 -10 11 (The dilution range of each round in the screening process is: the amplified phage culture supernatant 10 9 -10 11 ; unamplified panning isolate 10 2 -10 4 ). When the bacterial culture reached mid-log phase, divide into 200 μl aliquots in microcentrifuge tubes, one for each phage dilution. Add 10 μl of diff...

Embodiment 3

[0118] Example 3: Construction of a cell-penetrating peptide prokaryotic expression library

[0119] The polypeptide DNA sequence in the prokaryotic expression library of the present invention is derived from the total DNA of a random cyclic heptapeptide library screened by phage display technology. By comparing the recovery rate and enrichment factor of phage random heptapeptide library by four rounds of whole-cell screening, we found that the enrichment factor reached the maximum after the third round of screening. The decline of the enrichment factor in the fourth round suggests that the screening has approached saturation, and continued screening may lead to a decrease in diversity and loss of data. Therefore, the phage DNA extracted from the third round of screening was selected to construct the prokaryotic expression library of Hela cell-penetrating peptides. This step is a key control point that needs to be considered emphatically in the establishment of this method. ...

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Abstract

The invention relates to a method for constructing an expression library of cell penetrating peptide by utilizing bacteriophage display technology. The method comprises the following steps of adoptingthe bacteriophage display technology, combining with a whole-cell screening mode, and screening polypeptide with cytomembrane penetrating activity; then constructing pronucleus expression library ofcell penetrating peptide by utilizing the screened cell penetrating peptide; selecting and using a Ph.D.-c7c bacteriophage display peptide library, inserting the gene sequence of random heptapeptide into a bacteriophage capsid protein coding gene pIII, and enabling an expression product, which is corresponding to the random heptapeptide, and the capsid protein terminal of bacteriophage to form fusion protein, so as to form a combinatorial library. The positive cloning efficiency of the library constructed by the method is far higher than that of a random polypeptide library, the time period required for screening and verification is greatly shortened, and the method has the advantage of high flux.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to phage display, cell culture technology, prokaryotic expression library construction and molecular fluorescence technology, and specifically to a method for screening cell-penetrating polypeptides using phage display technology and using the screened polypeptides to construct a prokaryotic expression library, thereby Methods for validating and comparing the penetration capabilities of peptides. technical background [0002] The cell membrane is a semipermeable biological membrane, whose characteristics constitute the basis for the selective exchange of substances inside and outside the cell, and thus play a key role in maintaining the survival and normal function of cells. However, due to the existence of this natural effective barrier, some biomacromolecules and sensing molecules that have been proven to have good therapeutic effects and disease diagnostic value, such as non-fa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12N15/70C40B50/06
CPCC07K7/06C12N15/70C40B50/06
Inventor 姜勇刘芸罗海华
Owner SOUTHERN MEDICAL UNIVERSITY
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