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Optimized target object capturing system based on bacterial cell surface display system

A surface display system and bacterial cell technology, applied in the field of bioengineering, can solve the problems of cumbersome operation of the expression system, separation and acquisition of viral capsid proteins and receptors, etc., and achieve the effects of avoiding background interference, shortening the recovery cycle, and easy separation

Active Publication Date: 2017-10-20
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression systems of the two are cumbersome to operate and it is difficult to separate the complex of viral capsid protein and receptor (adsorption medium) from complex systems.

Method used

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  • Optimized target object capturing system based on bacterial cell surface display system
  • Optimized target object capturing system based on bacterial cell surface display system

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Construction of prokaryotic expression plasmid vector for inducible surface display virus capsid protein:

[0075] The artificially synthesized inaQn-TB fusion gene fragment, whose sequence is shown in SEQ ID No: 5, was ligated into the prokaryotic cloning vector pUC57, and the ligated product was transformed into Escherichia coli DH5α by the heat shock method. Positive clones were screened on the LB plate, and the single colony picked out was cultured and the plasmid was extracted from it to obtain the recombinant plasmid pUC57-inaQn-TB.

[0076] Use Nco I / EcoR I and Bgl II / EcoR I to perform double enzyme digestion on pET28a-inaQn-P(GII.4), then use Nco I / Bgl II to double enzyme digest pUC57-inaQn-TB, and recover About 5.3kb, 960bp, 543bp DNA fragments. Put the above three fragments in T 4 Ligation is carried out under the action of DNA ligase, and the product after enzyme ligation is transformed into Escherichia coli DH5α. Positive clones were screened on LB plates...

Embodiment 2

[0078] Expression of prokaryotic expression plasmid of inducible surface display viral capsid protein and functional identification of surface display system:

[0079] The prokaryotic expression plasmid pET28-inaQn-TB-P (GII.4) prepared in Example 1 was transformed into Escherichia coli BL21, carrying the prokaryotic expression plasmid pET28-inaQn-TB-P (GII. 4) Escherichia coli BL21 was named IL-2.4P. Pick a single colony and inoculate it in LB medium containing 100 μg / mL kanamycin. After culturing at 37°C for 12h to 14h, inoculate it in a fresh medium with 1.0% inoculation amount, and continue shaking culture until OD 600nmWhen it is 0.6, add IPTG with a final concentration of 0.4mmol / L, and continue shaking culture at 26°C for 16h. Centrifuge at 5000rpm for 10min at 4°C to collect the bacteria, then resuspend the bacteria in sterile PBS buffer with pH 7.2, OD 600nm Adjust to 1.0 and store at 4°C for later use.

[0080] Such as figure 1 As shown, by SDS-PAGE detection of ...

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Abstract

The invention discloses an optimized target object capturing system based on a bacterial cell surface display system, namely, the optimized target object capturing system comprises the following steps: inserting a segment of coding genes of adaptor protein between a virus capsid protein coding gene and a coding gene inaQn which is responsible for transmembrane, transfer and anchoring and is in an N-terminal structure domain of ice-nucleation protein, and establishing recombinant plasmid of inducible surface display virus capsid protein after verifying; transforming host bacteria, thus obtaining an updated bacterial cell surface display system of capsid protein; displaying virus capsid protein on the surface of escherichia coli cells through inducible expression, thus obtaining an optimized virus receptor capturing system. Compared with a traditional virus receptor purification and analysis method, the bacterial cell surface display system has the advantages that the cost of a reagent is low, the operation is simple, the experimental period is short, requirements on experimental equipment are low, a virus receptor can be easily separated, background interference is reduced, and the like; an updated virus receptor capturing system lays a solid foundation for efficiently discovering new virus receptors, and meanwhile, a new thought for protein purification and concentration is also provided.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a target capture system, in particular to an optimized target capture system based on a bacterial cell surface display system, such as a virus receptor or a specific adsorption ligand. 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 viral receptors mainly relies on co-immunoprecipitation methods and DNA-mediated transformation; then the viral receptors are verified by a large number of virus particles cultured and enriched in vitro; finally, the components and structures of viral receptors are obtained. However, there are no mature in vitro propagation systems for many viruses, and these methods are not practical. That is, the lack of a separation system for virus adsorption media is the main bottleneck hindering the in-depth study of virus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C07K1/14C12R1/19
CPCC07K1/14C07K14/005C07K14/21C07K2319/00C12N15/70C12N2770/16022
Inventor 王大鹏许茜倪培恩刘丹蕾殷玉洁史贤明
Owner SHANGHAI JIAO TONG UNIV
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