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Anchoring polypeptide taking basic amino acid as anchoring end and application of anchoring polypeptide

An acidic amino acid and amino acid technology, applied in peptides, fluorescence/phosphorescence, instruments, etc., can solve the problems of low cysteine ​​loading efficiency and time-consuming, and achieve excellent protection performance, less dosage, and enhanced stability.

Inactive Publication Date: 2018-12-11
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to improve the problem of low cysteine ​​loading efficiency and time-consuming in the traditional method, the present invention selects basic amino acids to form the anchor end of the polypeptide to replace the traditional pentapeptide CALNN, and under the mediation of the anchor polypeptide, realizes In order to quickly, efficiently and controllably load peptides or DNA on gold nanoparticles, and enhance the stability of gold nanoparticles

Method used

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  • Anchoring polypeptide taking basic amino acid as anchoring end and application of anchoring polypeptide
  • Anchoring polypeptide taking basic amino acid as anchoring end and application of anchoring polypeptide
  • Anchoring polypeptide taking basic amino acid as anchoring end and application of anchoring polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Design of anchoring polypeptide

[0042] Such as figure 1 As shown, three kinds of polypeptide sequences (Sequence 1, Sequence 2, Sequence 3) with basic amino acids (arginine, lysine, histidine) as anchor ends were designed, and fluorescent groups were modified on their protected ends 6-carboxyfluorescein, the three polypeptide sequences and their modified 6-carboxyfluorescein are expressed as follows:

[0043] serial number

Polypeptide sequences anchored by basic amino acids

Polypeptide modified with 6-carboxyfluorescein

sequence 1

RRFPDD

RRFPDD-FAM

sequence 2

KKFPDD

KKFPDD-FAM

sequence 3

HHFPDD

HHFPDD-FAM

[0044] (2) Preparation of gold nanoparticles (AuNPs)

[0045] Add 100mL concentration of 0.01% (w / w) chloroauric acid aqueous solution into the flask and heat to boiling, then add 1.5mL trisodium citrate aqueous solution (33.8mM) into it rapidly under vigorous stirring, when the color of the s...

Embodiment 2

[0048] Example 2: Optimization of anchoring polypeptides

[0049] (1) Selection of anchor terminal amino acids

[0050] Fluorescence signals at different time points of the three AuNPs-polypeptide-FAM dispersions prepared in Example 1 were obtained by a fluorometer, and fluorescence kinetic curves were prepared. Due to the fluorescence quenching effect of gold nanoparticles, the most efficient basic amino acids can be screened out according to the fluorescence kinetic changes of the three AuNPs-polypeptide-FAM dispersions. The result is as figure 1 As shown, arginine (R) among the three basic amino acids can bring faster and stronger fluorescence signal changes, that is, bring higher polypeptide loading efficiency. Therefore, we choose arginine as the anchor end of the polypeptide to promote the modification efficiency of the polypeptide.

[0051] (2) Selection of the number of amino acids

[0052] In addition, eight kinds of polypeptide sequences were designed and compared ...

Embodiment 3

[0067] (1) The anchor polypeptide RRFPDD connected with the target DNA, namely DNA-RRFPDD, was dissolved in NaH 2 PO 4 -Na 2 HPO 4 In the buffer solution (pH=7.4), the concentration of DNA-RRFPDD was 50 μM; then it was added to the AuNPs-phosphate dispersion, incubated at 37°C for 100 seconds and then centrifuged three times to remove excess DNA-RRFPDD, and the anchored polypeptide mediated Guided DNA-gold nanoparticles.

[0068] (2) Investigation of biological activity:

[0069] The hydrolysis performance of the polypeptide: select a target polypeptide with a fluorescent dye (sequence 26: RCFRGGDD, and load it on the gold nanoparticles by anchoring the polypeptide RRFPDD. After 100 seconds of loading, centrifuge and resuspend, add 100nM pancreatic Protease, detect fluorescence after incubation for 30 minutes. Through the change of fluorescence signal brought by enzymatic hydrolysis, verify whether its biological activity exists. Then by adjusting the modification ratio of...

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Abstract

The invention discloses an anchoring polypeptide RRFPDD. The aim of rapidly loading is achieved through carrying out anchoring by adopting basic amino acid instead of the traditional cysteine; acidicaspartic acid is used, so that a large number of negative charges are provided, and the protective property of the anchoring polypeptide to gold nanoparticles is enhanced; the self-assembly of the polypeptide is promoted through regulating and controlling the kind of hydrophobic amino acids. A biological detection reagent can be obtained through connecting a target probe to a protective end of theanchoring polypeptide and carrying out culture on the target probe and the gold nanoparticles in a phosphate buffer solution with the pH of 7.4 for 100 seconds at the temperature of 37 DEG C; the biological detection reagent can be used for detecting a concentration of a target detected substance by a standard curve method, and particularly, when a biological detection reagent prepared by takinga polypeptide with the sequence of FYSHSFHENWPS as a target probe is used for detecting myocardial troponin, the linear range is 0.05ng / mL to 500ng / mL, and the detection limit can reach 0.45ng / mL.

Description

technical field [0001] The invention relates to an anchor polypeptide with basic amino acid as the anchor terminal and a method for quickly, efficiently and controllably loading polypeptide or DNA on gold nanoparticles. Background technique [0002] The technology of loading biomolecules (such as peptides, DNA, etc.) on the surface of gold nanoparticles has greatly promoted the rapid development of nanobiotechnology, biosensors, and medical diagnosis. For this reason, a large number of applications have emerged, such as biosensing, disease diagnosis, and drug separation. [0003] A common loading method is to use the pentapeptide CALNN as the anchor polypeptide, and modify it by linking the polypeptide or DNA at the tail and using the sulfhydryl group in the cysteine ​​to bond to the nanoparticle. Not only that, the asparagine in CALNN has a large amount of negative charges, which can maintain the stability of gold nanoparticles during the loading process. Therefore, this ...

Claims

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Application Information

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IPC IPC(8): C07K7/06G01N33/68G01N21/64
CPCC07K7/06G01N21/6428G01N33/68G01N2021/6439G01N2333/4712
Inventor 胡继明李昕翼周晓东
Owner WUHAN UNIV
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