Phasmid display carrier pCANTAB5L

A phagemid and carrier technology, applied in the field of bioengineering, can solve the problems of reduced yield of recombinant phage, less number of endonuclease sites, inconvenient genetic engineering operation, etc., to expand the capacity of the phage display peptide library, which is beneficial to the activity The effect of maintaining and improving cloning efficiency

Inactive Publication Date: 2004-06-30
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when pCANTAB5X vector and other phagemid vectors are used to display exogenous polypeptides, since the PIII coat protein of the phage is directly fused with the displayed polypeptide, the PIII protein will affect the normal folding and spatial conformation of the exogenous protein, which is not conducive to maintaining The original activity and function of the displayed protein; similarly, foreign proteins will also have an impact on PIII protein molecules, because 5 PIII protein molecules are aggregated together, although there are wild-type PIII molecules in the 5 PIII proteins, but When exogenous proteins with large molecular weights are displayed, because the PIII molecules are directly connected to the foreign

Method used

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  • Phasmid display carrier pCANTAB5L
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  • Phasmid display carrier pCANTAB5L

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0048] Example 1: Using the recombinant phagemid vector pCANTAB5L to display a library of random variants of recombinant human lymphotoxin (rhLT)

[0049] In order to confirm whether the recombinant phagemid vector pCANTAB5L can correctly display the functional protein variant library, the recombinant human lymphotoxin (rhLT) random variant library cloned on the pGEM-T easy vector was digested with Xba I and Kpn I and directed Clone in the multiple cloning site of pCANTAB5L to construct a recombinant phage library displaying random variants of rhLT. The specific operation process is as follows:

[0050] (1) Construction of recombinant phage displaying a library of rhLT random variants

[0051] The rhLT / pGEM-T easy plasmid contains the recombinant human lymphotoxin derivative rhLT random variant library gene, the library capacity is 1×10 6The recombinant human lymphotoxin expressed by the rhLT gene lacks 23 amino acids at the N-terminal, and contains an Xba I restriction site at th...

Example Embodiment

[0057] Example 2: Use the recombinant phagemid vector pCANTAB5L to display the macromolecular polypeptide-Staphylococcus protein A derivative (Mu-protein A):

[0058] In order to test whether pCANTAB5L can correctly display macromolecular peptides, the gene of Staphylococcus protein A derivative (Mu-protein A) was digested with Xba I and Kpn I from pGEM-T easy vector and cloned into the multiple cloning site of pCANTAB5L. In point, construct a recombinant phage displaying Mu-protein A. The specific operation process is as follows:

[0059] (1) Construction of recombinant phage displaying Mu-protein A:

[0060] Staphylococcal protein A (Mu-protein A) consists of 360 amino acids and contains 5 antibody binding domains. Mu-protein A / pGEM-T easy plasmid is cloned with Mu-protein A gene, and the 5'end contains the Xba I restriction site and the 3'end contains the Kpn I restriction site. Mu-protein A / pGEM-T easy plasmid was recovered with the Xba I and Kpn I double digestion fragment wi...

Example Embodiment

[0081] Example 3: Use the recombinant phagemid vector pCANTAB5L to display human interferon (hIFNαA-2b):

[0082] In order to compare whether the recombinant phagemid vector pCANTAB5L containing [G4S]3 polypeptide linker can maintain its biological activity better than the original pCANTAB5X phagemid when displaying functional proteins, human interferon αA-2b (hIFNαA-2b) Clone into the multiple cloning site of pCANTAB5L, construct a recombinant phage displaying hIFNαA-2b, and use pCANTAB5X vector to display a recombinant phage displaying hIFNαA-2b [Wu Xiaolan et al., Journal of Second Military Medical University, 2000, 23(4):403] Comparison of interferon activity. The specific operation process is as follows:

[0083] (1) Construction of recombinant phage displaying hIFNαA-2b:

[0084] The hIFNαA-2b / pGEM-T easy plasmid contains the human interferon αA-2b gene, and the interferon encoded by the gene consists of 166 amino acids. The 5'end of the hIFNαA-2b gene contains the Xba I res...

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Abstract

The invention is a new phage mid display vector pCANTAB5L, applied to the phage for displaying the exotic random polypeptide. It inserts the linker fragment Xba I-Stu I-Sal I-Kpn I-[G4S] 3-Not I between SfiI and NotI endonuclease cutting sites of pCANTAB5L to obtain the vector. The vector provides 5 common endonuclease cloning sites Sfi I, Xba I, Stu I, Sal I and Kpn I, which makes exotic gene oriented cloning convenient and improves the cloning efficiency, beneficial to enlarge the polypeptide library capacity displayed by the phage; the vector introduces flexible polypeptide linker gene [G4S] 3 composed of 15amino acids between the phage PIII protein gene and the exotic display protein gene, and properly uses Escherichia coli rare codon, so as to reduce the mutual interference of the displayed exotic and PIII proteins and maintain the function and activity of the displayed exotic and PIII proteins. It is applied to displaying various random peptide library, functional target protein, functional target protein variant library and large molecular-weight (>300 amino acids) functional protein.

Description

Technical field: [0001] The invention relates to the technical field of bioengineering, and is a novel phagemid display carrier pCANTAB5L used in phage display of exogenous random polypeptides. Background technique: [0002] The so-called phage display technology is a technology that uses phage to fuse foreign polypeptide genes with phage structural protein genes and then use it to display foreign polypeptides on the surface of phage. Because phage has the characteristics of simple structure, small size, large number per unit volume, easy replication, and suspendability in liquid, phage display technology has been widely used in the study of molecular interactions. These mainly include the use of affinity selection to capture target receptor sequences from display peptide libraries, epitope identification or epitope simulation, identification of new receptors and natural ligands, selection of DNA binding proteins, search for new drugs, and provide vaccine development and Ne...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/70
Inventor 潘卫沈毅君刘彦君戚中田
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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