Peptide of specific combined ricin, its coding nucleic acid and method of detecting and inhibiting ricin

A technology of ricin and polynucleotides, which is applied in botany equipment and methods, biochemical equipment and methods, peptide sources, etc., can solve the problems of restricting wide application, strong toxicity, and strong toxicity of ricin

Inactive Publication Date: 2005-03-09
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to its strong toxicity, its wide application is limited. For this reason, research on ricin modifications is also being carried out in order to reduce the toxicity an

Method used

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  • Peptide of specific combined ricin, its coding nucleic acid and method of detecting and inhibiting ricin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Screening of ricin-specific binding peptides

[0092] method:

[0093] a. Coating: ricin (RC) (provided by the fifth room of the Sixth Academy of Military Medical Sciences) was coated on an enzyme-linked plate with 10 μg / 100 μl / well of carbonate buffer solution with pH=8.0, and overnight at 4°C;

[0094] b. Sealing: remove the coating solution in the well, seal the coated well and the blank control well with 1% gelatin, 300 μl / well, room temperature for more than 1 hour;

[0095] c. Washing: remove the blocking solution and wash 6 times with TBS-T (0.1% Tween-20);

[0096] d. Binding: Dilute the phage with TBS-T (0.1% Tween-20), add 100 μl (containing 2×10 11 pfu), incubated with gentle shaking at room temperature, the time for the first cycle is 90min, the second cycle is 60min, and the third cycle is 45min;

[0097] e. Cleaning: remove unbound phage (ph.D.-12phage Peptide Library displayed on the surface of phage surface capsid protein III, purchased from...

Embodiment 2

[0102] Example 2 ELISA detection specific binding peptide

[0103] a. Coating plate: RC was diluted with carbonate buffer (pH=8.0), coated in an enzyme-linked plate, 1 μg / 100 μl / well, overnight at 4°C;

[0104] b. Sealing: 10% skimmed milk powder (diluted in PBS) was added to the experimental well and the blank control well at 200 μl / well, and incubated at 37°C for 1.5h;

[0105] c. Cleaning: wash 5 times with PBS-0.3% T and shake dry;

[0106] d. Binding: add 1 μg / well of phage (the phage after three rounds of screening are cultivated overnight in a culture plate containing host bacteria, then pick and amplify the plaque) 1 μg / well, dilute with PBS, and shake gently at room temperature for 1 hour;

[0107] e. Cleaning: wash 5 times with PBS-0.3% T and shake dry;

[0108] f. Add the primary antibody: Dilute the anti-M13 polyanti-rabbit serum (prepared by the third and eighth rooms of the Military Medical College) at 1:5000, 100 μl / well, and incubate at room temperature for 1...

Embodiment 3

[0125] Example 3 Sequencing Results of Specific Binding Peptides

[0126] The specific binding peptide was sequenced by Shanghai Boya and Shanghai Shenyou Biotechnology Company. The sequence is as follows.

[0127] 1 L L I R Q P (SEQ ID NO: 1)

[0128] 7L R M S P S (SEQ ID NO: 2)

[0129] 8 T P R H I I HQR (SEQ ID NO: 3)

[0130] 10 R S P N I R (SEQ ID NO: 5)

[0131] 17 Q I I R Q S N N M (SEQ ID NO: 7)

[0132] 22 R N M S S L (SEQ ID NO: 8)

[0133] 23 R T Q I Q (SEQ ID NO: 9)

[0134] 26 R P R I P K (SEQ ID NO: 10)

[0135] 27 R I M T N R T L T (SEQ ID NO: 11)

[0136] 9 I N R Q R S H I (SEQ ID NO: 4)

[0137] 16 S R Q R H L R NR (SEQ ID NO: 6)

[0138] 29 I L Q S (SEQ ID NO: 12)

[0139] Amino acids K and R, M and L have certain similarities in structure and properties. We can get two groups of conserved sequences, one group is RRX, and the other group is LLX or LLLX (X represents an...

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Abstract

The invention discloses peptide of specific banded ricin, nucleic acid for encoding the same, and method for detecting and restraining the ricin. Preferably, the peptide of the invention has all showed amino acid sequences of SEQ ID NO: 1 to 12.

Description

technical field [0001] The present invention mainly relates to peptides specifically binding to ricin, nucleic acids encoding them and methods for detecting and inhibiting ricin. Background technique [0002] Ricin (RC) is a plant protein extracted from castor seeds of the Euphorbiaceae plant. It is a member of the plant lectin kingdom, most of which bind carbohydrates and are toxic. These lectins can be divided into two types, one is RIP type I (ribosome-inactivating protein type I), which is a single-chain protein, including trichosanthin, pokeweed antiviral protein, etc.; the other is RIP type II , including: abrin, ricin, mistletoe, etc. The A chain of RIP type II is similar in structure and function to RIP type I, and both have highly conserved regions in sequence analysis. Ricin belongs to RIP type II and consists of two chains, A and B. The A chain has 263 amino acids and has N-glycosidase activity, which can hydrolyze the adenine at position 4323 of eukaryotic 28...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63G01N33/68
Inventor 王亚丽李前王玉霞邵宁生
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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