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A method for displaying foreign protein macromolecules on the surface of t4 bacteriophage using the intracellular synchronous expression method

A technology of exogenous protein and phage, which is applied in the fields of biochemistry and molecular biology, can solve the problems of overexpression of exogenous recombinant protein, solution solubility of recombinant protein and separation and purification efficiency, so as to avoid protein separation and purification Process, manpower saving effect

Active Publication Date: 2014-10-29
江苏愚公生命科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process will face: 1) the expression level of the overexpression of the exogenous recombinant protein; 2) the solution solubility and separation and purification efficiency of the recombinant protein; Combining efficiency issues

Method used

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  • A method for displaying foreign protein macromolecules on the surface of t4 bacteriophage using the intracellular synchronous expression method
  • A method for displaying foreign protein macromolecules on the surface of t4 bacteriophage using the intracellular synchronous expression method
  • A method for displaying foreign protein macromolecules on the surface of t4 bacteriophage using the intracellular synchronous expression method

Examples

Experimental program
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Embodiment 1

[0025] Example 1. A method for displaying foreign protein macromolecules on the surface of T4 phage using synchronous intracellular expression. The steps are as follows:

[0026] (1) The construction of the expression plasmid of the Hoc protein with the natural regulatory region of T4 phage or the recombinant protein fused with the Soc protein and foreign protein:

[0027] (1) Fusion of the gene coding region of the Hoc protein or Soc protein with the gene coding region of the foreign protein to be displayed, and adding the natural regulatory region of T4 phage to control the expression of Hoc protein or Soc protein in the upstream of the sequence; foreign protein selection From any protein; after fusion, a gene sequence encoding Hoc protein or a recombinant protein fused with foreign protein with regulatory sequence is formed; plasmids containing this sequence will be synchronized in the process of infection and assembly of T4 phage in E. coli cells Express foreign recombinant pro...

experiment example

[0034] Experimental examples, the following two examples illustrate the technical solution of the present invention.

[0035] Two naturally folded, functionally complete foreign proteins: GG protein and gal protein are fused with Soc protein and Hoc protein respectively to form recombinant protein, which is displayed on the capsid of T4 phage by synchronous intracellular expression.

[0036] (1) Construction of the expression plasmid of the recombinant protein fused with Soc protein and gal protein:

[0037] (1) Amplify the gene sequence of the Soc protein by PCR, including its promoter region (the gene sequence of the Soc protein promoter region is: tataaataatcatgtaatttaaataaaggagaattac). Primer 1 is the first 18 bases of the gene sequence in the promoter region of the Soc protein. Primer 2 is the reverse complement of the following sequence: the last 21 bases of the coding region of the Soc protein gene remove the stop codon, plus the first 18 bases of the coding region of the gal...

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Abstract

The invention discloses a method for showing a foreign protein macromolecule on the surface of a T4 bacteriophage by using an intracellular synchronous expression method. The method for showing the foreign protein macromolecule on the surface of the T4 bacteriophage comprises the following steps that an expression plasmid of recombinant protein fused by Hoc (Highly-antigenic Outer Capsid) protein or Soc (Small Outer Capsid) protein with a natural T4 bacteriophage control area and foreign protein is constructed; the expression plasmid of the foreign recombinant protein is transformed into an escherichia coli cell; the escherichia coli cell containing the expression plasmid of the foreign recombinant protein is infected by an Hoc protein and Soc protein deleted T4 bacteriophage virus strain; T4 bacteriophage particles generated in the infection step and showing the foreign recombinant protein are separated; and the foreign recombinant protein shown on capsid of the obtained T4 bacteriophage particles is detected. The method does not require overexpression of the foreign recombinant protein, avoids a technical difficulty that large-amount overexpression of the foreign recombinant protein is required in an in-vitro showing method, does not require separation or purification of the foreign recombinant protein, and avoids a time-consuming and expensive protein separation and purification process.

Description

Technical field [0001] The present invention relates to the fields of biochemistry and molecular biology, in particular to a method for displaying foreign protein macromolecules on the surface of T4 phage using an intracellular synchronous expression method. Background technique [0002] There are two modified proteins on the surface of the capsid of T4 phage, namely Hoc protein and Soc protein. These two proteins are non-essential proteins, that is, deletion or modification of these two proteins will not affect the replication and amplification of T4 phage. Based on the characteristics of the two protein molecules, and through molecular biological means, foreign proteins (that is, non-T4 phage proteins) can be recombined with Hoc or Soc proteins and displayed on the surface of T4 phage. The technology of displaying foreign recombinant proteins on the surface of T4 phage through molecular biological means has shown great potential application value in vaccine development. [0003...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N7/01C12R1/92
Inventor 高嵩陶攀许恒皓刘伟尹欣程斌
Owner 江苏愚公生命科技有限公司
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