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Polypeptide random library and its construction method, and method for screening polypeptide capable of penetrating cell from the library

A technology for constructing methods and libraries, applied in the field of biomedicine, can solve problems such as no random library, and achieve the effect of wide use and simple screening method.

Inactive Publication Date: 2006-02-15
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is also no report on the method of screening random libraries to obtain CPP

Method used

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  • Polypeptide random library and its construction method, and method for screening polypeptide capable of penetrating cell from the library
  • Polypeptide random library and its construction method, and method for screening polypeptide capable of penetrating cell from the library
  • Polypeptide random library and its construction method, and method for screening polypeptide capable of penetrating cell from the library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Random library construction.

[0064] The first step: construction of pET-14bMCS vector. This step is to add four enzyme cutting sites Apa I (GGGCCC), Kpn I (GGTACC), Sma I (CCCGGG) and Not I (CGGCCG) between the multiple cloning sites Nde I and Xho I of the pET-14b vector. The method of introducing mutations by PCR was used to complete the vector construction. The upstream primer was 5′-GGGCGGCCGCTCGAGGGATCCGGCTGCTAACAAAGCCG-3′; the downstream primer was 5′-GGGGGTACCGGGCCCCATATGGCTGCCGCGCGGCACCAG-3′. Using the pET-14b plasmid as a template, a PCR reaction was performed with Pyrobest high-fidelity polymerase (TaKaRa Company), and the reaction conditions were: 95° C. for 30 s; 56° C. for 30 s; 72° C. for 5 min. Perform agarose gel electrophoresis on the PCR reaction solution, and cut the gel to recover the target DNA fragment. According to the operation guide of TaKaRa MutanBEST Kit, mix 17 μl of the target fragment, 2 μl of 10×Blunting Kination Buffer, 1 μl of Bluntin...

Embodiment 2

[0091] Random library construction.

[0092] The first step: construction of pET-14bMCS vector. Its construction method is as described in the first step of Example 1.

[0093] The second step: construction of pET-14bMCStop vector. Its construction method is as described in the first step of Example 1.

[0094] The third step: construction of pET-14bMCStop / EGFP vector. EGFP was subcloned into the pET-14bMCStop vector. The EGFP coding gene is obtained by using the pEGFP-C2 vector as a template, amplified by PCR technology, and adding a Kpn I site at the 5' end of the fragment, and adding an Xho I site at its 3' end.

[0095] Step 4: construction of random peptide library. Its construction method is as described in the first step of Example 1.

[0096] Step 5: transform to obtain a random expression library for screening cell-penetrating polypeptides. Its method is as described in the fifth step of Example 1.

Embodiment 3

[0098] Construction of pET-14b / Tat-EGFP.

[0099] The first step: construction of pET-14b / Tat vector. Its construction method is as described in the first step of Example 1.

[0100] The second step: construction of pET-14b / Tat-EGFP vector. EGFP was subcloned into the pET-14b / Tat vector. The EGFP coding gene is obtained by using the pEGFP-C2 vector as a template, amplified by PCR technology, and adding a Kpn I site at the 5' end of the fragment, and adding an Xho I site at its 3' end.

[0101] The expression of pET-14b / Tat-EGFP and the positive result detection of the expressed protein His-Tat-EGFP penetrating the cell membrane:

[0102] Escherichia coli BL21 (DE 3), ampicillin plate selection, incubated overnight at 37°C, picked a single clone in the next morning and added 1.6ml LB / Amp + (100μg / ml) culture medium, use 48-well deep-well plates to shake at 37°C until the OD600 is about 0.6, then add a final concentration of 1mmol / LIPTG to the bacterial solution to induce p...

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Abstract

The invention provides a random peptide library, wherein any one fusion protein in the random peptide library comprises a label for purifying the fusion protein, a labeling protein and random polypeptide, wherein the labeling protein is linked to the end of the carboxyl of the label, the random polypeptide is linked to the end of the carboxyl of the labeling protein. The invention also provides a method for constructing the random polypeptide library and a method for screening cells from the library to penetrate polypeptides.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a polypeptide library, a construction method and its application, in particular to a random library for screening cell-penetrating polypeptides, a construction method and a method for screening cell-penetrating polypeptides from the library. technical background [0002] The cell membrane is a semipermeable barrier between the cell and the extracellular environment, with selective permeability so that the cell maintains a constant internal environment. In molecular biology research, gene therapy and pharmaceutical industry, macromolecules such as proteins, peptides, nucleotides and drugs need to be introduced into cells to play a role. Due to the selective permeability of the cell membrane, it is difficult for the above-mentioned substances to pass through the cell membrane to reach the corresponding drug target in the cell. Existing techniques for intracellular transport thro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00G01N21/64C12N15/62C12N15/63C12P21/00
Inventor 姜勇刘亚伟刘瑜刘靖华邓鹏李海玉
Owner SOUTHERN MEDICAL UNIVERSITY
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