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Virus receptor capture system based on bacterial surface display system

A surface display system and virus technology, applied in the field of bioengineering, to achieve the effects of easy separation, shortening the recovery cycle and reducing costs

Inactive Publication Date: 2016-01-20
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, up to now, there is no report on the use of bacterial cell surface display system for virus receptor analysis

Method used

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  • Virus receptor capture system based on bacterial surface display system
  • Virus receptor capture system based on bacterial surface display system
  • Virus receptor capture system based on bacterial surface display system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Cloning of ORF2 and its 3' end fragment and inaQn gene fragment

[0054] A pair of specific primers (called P1 and P2) were designed according to the human norovirus main capsid protein coding gene (ORF2) sequence published by GenBank of the National Center for Biotechnology Information (NCBI), according to the inaQ published in GenBank. A pair of specific primers (referred to as P3 and P4) were designed, and RNA from clinical samples of Norovirus diarrhea and genomic DNA of Pseudomonas syringae were respectively used as templates for RT-PC or PCR amplification to obtain ORF2 gene fragment and inaQn gene fragment. Using ORF2 as a template, design a pair of specific primers (called P5 and P6) and carry out PCR amplification to obtain the 3' end gene fragment of ORF2 (ie, the P gene fragment). Specific primer information is as follows:

[0055] P1 (5'→3'): GGA AGATCT ATGAAGATGGCGTCG

[0056] P2(5'→3'): CCG GAATTC TTATAAAGCACGTCTG

[0057] P3(5'→3'): CCATGG ATGGA...

Embodiment 2

[0067] Construction of Prokaryotic Expression Plasmid Vector of Inducible Surface Display Virus Capsid Protein

[0068] The ORF2 gene fragment obtained above, the 3' end gene fragment of ORF2 (i.e. the P gene fragment) and the inaQn gene fragment were respectively connected to the pMD19-T vector, and the ligated product was transformed into E. After the white spot screening, single white spot colony was picked, and the picked single colony was cultured to extract plasmids therefrom to obtain recombinant plasmids pMD19-ORF2, pMD19-P and pMD19-inaQn.

[0069] Double enzyme digestion of pMD19-ORF2 and pMD19-P with BglII / EcoRI, double enzyme digestion of pMD19-inaQn with BglII / NcoI, and 1.5% agarose gel electrophoresis analysis respectively, the recovery is about 1623bp, 960bp and a 525bp DNA fragment, and carry out EcoRI / NcoI double enzyme digestion on the inducible expression plasmid pET-28a. The above-mentioned recovered gene fragments were divided into two groups (inaQn+ORF2+...

Embodiment 3

[0071] Expression of prokaryotic expression plasmids for inducible surface-displayed viral capsid proteins

[0072]The prokaryotic expression plasmids pET28-inaQn-ORF2 and pET28-inaQn-P carrying the inducible surface display virus capsid protein in Example 2 were respectively transformed into Escherichia coli BL21 to obtain a positive clone JTU-2.4 (containing the pET28-inaQn-ORF plasmid) and JTU-2.4P (containing the pET28-inaQn-P plasmid), pick a single colony of JTU-2.4 and JTU-2.4P, inoculate in LB medium containing 100 μg / mL Kan, and culture at 37°C for 12 to 14 hours, Inoculate in fresh medium with 1% (V / V) inoculum, continue shaking culture to OD 600 When it is 0.6, add IPTG with a final concentration of 0.4mmol / L, and continue shaking culture at 26°C for 24 hours. Collect the cells by centrifugation at 5000×g for 10 min at 4°C, then resuspend the cells in sterile PBS buffer with pH 7.2, OD 600 Adjusted to 1.0, stored at 4°C for later use.

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Abstract

The invention discloses a method for preparing a virus receptor capture system based on a bacterial surface display system. The method comprises steps as follows: a capsid protein coding gene segment of a virus is fused with a coding gene segment of a structural domain of a coding ice nucleating protein N terminal and implanted into an inducing type prokaryotic expression plasmid, a recombinant inducing type expression plasmid is obtained and transformed into host bacteria, and the bacterial surface display system of capsid protein of the virus is acquired; the capsid protein of the virus is displayed on the surface of the bacteria, and the virus receptor capture system of the virus is obtained. The virus receptor capture system prepared with the method doesn't adopt the complicated step of purification of the viral capsid protein, the cost for obtaining the viral capsid protein is greatly reduced, the recycling period of viral capsid and virus receptor compounds is substantially shortened, corresponding virus receptors in the environment are easier to separate, and the system has the benefits of saving and environmental protection. The system can be used for exploring a new receptor of the virus or analyzing and identifying components and structure of the virus receptor and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a virus receptor capture system based on a bacterial surface display system, in particular to a virus receptor capture system that cannot be cultured in vitro. Background technique [0002] Analysis and identification of viral receptors is a key step in exploring the mechanism of virus invasion into cells and host infection. At present, the analysis of virus receptors mainly relies on a large number of virus particles cultured and enriched in vitro, and the virus receptors are specifically adsorbed; then, the complexes of viruses and their receptors are obtained by ultra-high-speed centrifugation; finally, the complex The components and structures of virus receptors are obtained through the analysis and identification of substances, which are also used to explore new virus receptors. [0003] The classic viral receptor analysis method is mainly aimed at viruses that can...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21G01N33/68
Inventor 王大鹏李茜茜牛梦雅喻钱钱崔妍史贤明
Owner SHANGHAI JIAO TONG UNIV
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