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T4 bacteriophage surface exogenous protein high effective expression vector and establishing method thereof

A technology for expressing foreign proteins on the surface, applied in the field of high-efficiency expression vectors and creation of foreign proteins on the surface of T4 bacteriophage, can solve the problem of loss of functional activity and other problems

Inactive Publication Date: 2004-08-25
任兆钧
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, often only the primary structure of the target protein-polypeptide is expressed, and some foreign proteins expressed and displayed lose their functional activity due to the inability to fully express the spatial structure necessary for their biological activity.

Method used

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  • T4 bacteriophage surface exogenous protein high effective expression vector and establishing method thereof
  • T4 bacteriophage surface exogenous protein high effective expression vector and establishing method thereof
  • T4 bacteriophage surface exogenous protein high effective expression vector and establishing method thereof

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Embodiment

[0034] The methods for creating the surface expression display vectors of the three strains of T4 phage are as follows:

[0035] 1. The creation method of T4 phage SOC site surface expression display vector (ΦT4△Soc):

[0036] 1) Using the wild-type T4 phage as a template, and the plasmid P△Soc9.8 in the engineering bacteria E.coli

[0037] Carry out homologous recombination in CR63, use SOC antibody as a probe, and perform immunoprotein hybridization

[0038] The mutant T4 phage strain T4-△9.8Soc with 9.8kb deletion in the Soc gene region was screened out;

[0039] 2) The T4 phage mutant strain Eg326 (S12+, △IPI, Alt - , e - , denV - ) and T4

[0040] Phage T4-△9.8Soc recombination;

[0041] 3) Using engineering bacteria E.coli CR63 as indicator bacteria, cultivate overnight at 37°C, in lysozyme-dependent

[0042] The target carrier ΦT4 was screened out on the culture medium (this medium plus 50 μg / ml lysozyme)

[0043] △Soc.

[0044] 2. The creation method...

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Abstract

The present invention relates to a kind of T4 bacteriophage surface exogenous protein high-effective expression vector and its creation method. Said vector includes: (1) T4 bacteriophage SOC site surface expression showing vector phiT4 delta Soc; (2) T4 bacteriophage HOC site surface expression showing vector phiT4 delta Hoc; and (3). SOC-HOC double-site surface expression showing vector phiT4 delta Soc-delta Hoc. Said invention is a maximum genome (1.6X10 to the power 5 nucleotide) in the currently-known bacteriophages, one bacteriophage granule surface can simultaneously express and show two protein polypeptides whose quality sources are different on double-side (SOC and HOC), its copy number can be up to 1120, and the expressed exogenous protein space structure is unchangeable, and can retain its original biological activity. Said invention also provides its application.

Description

technical field [0001] The invention relates to the technical field of gene engineering protein expression, in particular to a high-efficiency expression vector of exogenous protein on the surface of T4 bacteriophage and its creation method. Background technique [0002] Since Professor SMITH of the United States proposed the pioneering concept of using phage surface expression to display proteins expressed by foreign genes in 1988, several phage vector varieties such as phage M13 and T7 have been applied in the laboratory and commercialized for ten years. above history. However, the surface expression and display vectors of these phage varieties have small phage genomes and small surface space, and the foreign proteins that can be packaged and expressed are limited to a dozen to dozens of amino acid polypeptides in length. The copy number is low, less than about 300 copies, and only one foreign peptide can be expressed at a single site on the surface of the phage. Moreove...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/33C12N15/62C12N15/86
Inventor 任兆钧
Owner 任兆钧
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