Phasmid display carrier pCANTAB5L

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...

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 proteins, the connection lacks flexibility and cannot rotate freely, and its huge spatial structure will also cause steric hindrance to the wild-type PIII protein, thereby affecting its infection activity, which reduces the yield of recombinant phage; moreover, pCANTAB5X only contains 3 cloning sites, Sfi I, XbaI and Not I, and there is only one Xba I in the commonly used endonuclease site, so it is the same as other commercialized phagemid vectors. There is a problem that the number of cloning sites, especially the number of commonly used endonuclease sites, is small, which brings inconvenience to genetic engineering operations, especially when constructing a random display peptide library, which limits the library capacity

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