Polystyrene affinity peptide and method of polystyrene affinity peptide for improving immobilized effect of antigen

A polystyrene, affinity technology, applied in the field of affinity peptide sequence PB-TUP, can solve the problems of loss of function, affect detection sensitivity and accuracy, loss of biological activity, etc., to avoid inactivation, improve immobilization effect, Effect of improving detection sensitivity

Inactive Publication Date: 2017-12-26
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is not only time-consuming and requires multi-step reactions to promote the combination of peptides and materials, but also the loss of biological activity due to changes in the spatial conformation of biomolecules during the coupling process.
In addition, the non-specific adsorption of polypeptides and proteins on the solid phase surface is disordered and has no directionality. Some proteins or polypeptides will lose their function due to conformational changes or active sites are covered, thus affecting the sensitivity and accuracy of detection or subsequent research.

Method used

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  • Polystyrene affinity peptide and method of polystyrene affinity peptide for improving immobilized effect of antigen
  • Polystyrene affinity peptide and method of polystyrene affinity peptide for improving immobilized effect of antigen
  • Polystyrene affinity peptide and method of polystyrene affinity peptide for improving immobilized effect of antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Screening for sequences with specific affinity to polystyrene microplates

[0035] experimental method:

[0036] 1. Biopanning procedure

[0037] 1. Blocking: 250 μL of 3% (w / v) BSA-TBS solution was added to each well to block the polystyrene microwell plate at 37° C. for 2 h.

[0038] 2. Incubation: After discarding the blocking solution and washing quickly, add 100 μL of the phage dodecapeptide library to the microwell plate and incubate at 37°C for 1 hour.

[0039] 3. Non-specific washing: the microwells were washed 5 to 15 times with 0.05% (v / v) Tween-TBS solution, and then washed 3 times with TBS.

[0040] 4. Specific elution: Discard the washing solution, add 150 μL of 100 mM HCl-Glycine (pH 2.2) solution for 10 min, and immediately add 9 μL of 2M Tris-HCl (pH 9.1) solution to neutralize the eluted phage.

[0041] 5. Amplification: Take 5 μL of this solution for phage titer determination and put the remaining solution into 25 mL of ER2738 bacterial solution in ...

Embodiment 2

[0061] Comparison of the binding ability of each phage clone to polystyrene material under the conditions of different blocking solutions and washing solutions in the enzyme-linked immunosorbent assay

[0062] experimental method:

[0063] 1. Preparation of solutions: Six different solutions of PBS, 0.05% PBST, TBS, 0.05% TBST, 3% BSA-TBS and 3% NFM-TBS were prepared as blocking and washing solutions, respectively.

[0064] 2. Blocking: Use the above six solutions to block the polystyrene microplate at 37°C for 2 hours.

[0065] 3. Incubation of Phage: 10 10 (pfu / mL) PB-TUP phage clone, phage dodecapeptide library, wild-type M13KE phage clone and irrelevant peptide phage clone D12 were used as the experimental group and the control group, and were added to each polystyrene microwell treated with different blocking solutions. , and incubated overnight at 4°C.

[0066] 4. Washing: wash the above six solutions as different washing solutions for 5 times respectively

[0067] 5...

Embodiment 3

[0071] Eliminates the possibility of false positives due to phage adsorption to proteins in the blocking solution skim milk

[0072] experimental method:

[0073] 1. Blocking: NFM-TBS with different concentrations (0.00% to 3.20%) was added to each well and blocked at 37°C for 2 hours.

[0074] 2. Incubation of Phage: Bring the titer to 10 10 (pfu / mL) PB-TUP phage clone, phage dodecapeptide library, wild-type M13KE phage clone and irrelevant peptide phage clone D12 were used as the experimental group and the control group. Incubate overnight at 4 °C.

[0075] 3. Washing: Wash the microwells 5 times with 0.05% TBST solution.

[0076] 4. Incubation and detection of secondary antibody: add secondary antibody to incubate and carry out the subsequent steps of ELISA detection in Example 1.

[0077] Experimental results:

[0078] The result is as figure 2 As shown, NFM-TBS blocking solution inhibited all phage clones and polystyrene in a concentration-dependent manner. The bi...

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Abstract

The invention discloses a polypeptide sequence specifically bind to polystyrene and a method for improving polypeptide and protein antigen for surface of a polystyrene material. By adopting a phage display random peptide library screening, the polypeptide PB-TUP which is subjected to affinity binding with polystyrene is obtained, an amino acid sequence is Val-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser; and a coding nucleotide sequence is GTG-CAT-TGG-GAT-TTT-CGG-CAG-TGG-TGG-CAG-CCT-TCT. The method has the following advantages that 1) then affinity peptide PB-TUP has strong affinity binding capability on a polystyrene carrier; 2) the affinity peptide PB-TUP and polypeptide or protein antigen are fusion, so that the immobilized effect of antigen can be effectively improved; 3) the affinity peptide PB-TUP can guide the fusion antigen to immobilize on the surface of polystyrene by an unified and ordered mode, the space conformation of the antigen is kept, and the inactivation of antigen active sites due to shielding can be avoided; and 4) the method can be used for diagnosis detection technologies, such as enzyme linked immunosorbent assay, biology, cytological detection, and biosensors and micro-array chips which take the polystyrene as a fixed carrier, and improves detection sensitivity.

Description

technical field [0001] The invention relates to an affinity peptide sequence PB-TUP, which can specifically bind polystyrene materials. And the method for improving the fixing effect of polypeptide and protein on the surface of polystyrene material by the peptide. Background technique [0002] The phage display technology was created by George P. Smith in 1985. By engineering the phage, the exogenous amino acid sequence can be displayed on the end of the capsid protein while ensuring its ability to infect and reproduce. Through at least three rounds of biological screening procedures of adsorption, elution, and amplification, phages with specific affinity for the target are enriched from many random phages. By sequencing the phage DNA, the nucleic acid sequence encoding the polypeptide bound to the target can be identified, and the amino acid sequence of the encoded polypeptide can be obtained. Phage display technology is not only widely used in basic research and drug dev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11G01N33/531G01N33/547
CPCC07K7/08G01N33/531G01N33/54353
Inventor 王旻邱郑强旭
Owner CHINA PHARM UNIV
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