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Vitellogenin peptide fragment tfVWD used for binding to tetrodotoxin, nucleotide sequence of vitellogenin peptide fragment tfVWD, polyclonal antibody of vitellogenin peptide fragment tfVWD and preparation method for vitellogenin peptide fragment tfVWD

A technology of vitellogenin and nucleotide sequences, which is applied in the fields of peptide preparation methods, botanical equipment and methods, biochemical equipment and methods, etc., to achieve the effect of low price, simple configuration and high purity

Pending Publication Date: 2020-07-03
FISHERIES RES INST OF FUJIAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no related report on the separation and purification of TTX-binding protein from Fugu chrysanthemum

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  • Vitellogenin peptide fragment tfVWD used for binding to tetrodotoxin, nucleotide sequence of vitellogenin peptide fragment tfVWD, polyclonal antibody of vitellogenin peptide fragment tfVWD and preparation method for vitellogenin peptide fragment tfVWD
  • Vitellogenin peptide fragment tfVWD used for binding to tetrodotoxin, nucleotide sequence of vitellogenin peptide fragment tfVWD, polyclonal antibody of vitellogenin peptide fragment tfVWD and preparation method for vitellogenin peptide fragment tfVWD
  • Vitellogenin peptide fragment tfVWD used for binding to tetrodotoxin, nucleotide sequence of vitellogenin peptide fragment tfVWD, polyclonal antibody of vitellogenin peptide fragment tfVWD and preparation method for vitellogenin peptide fragment tfVWD

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Embodiment 1

[0033] The present invention discloses a vitellogenin peptide tfVWD used for binding tetrodotoxin, which contains a polypeptide of the amino acid sequence shown in SEQ ID NO: 1 or an active fragment of the polypeptide.

[0034] The tfVWD domain exists in various proteins such as vitellogenin, and is a highly structured region, in which a pair of disulfide bonds are formed between the first and second conserved Cys.

[0035] The invention discloses a preparation method of vitellogenin peptide tfVWD for binding tetrodotoxin, comprising the following steps:

[0036] S1. Construction of a recombinant expression vector for the provitolin peptide tfVWD of puffer chrysanthemum

[0037] 1. Cloning of tfVWD gene sequence of puffer chrysanthemum vitellogenin

[0038] (1) First, in the NCBI database, search for the vitellogenin tfVWD domain (tfVWD) gene sequence of red-finned puffer and puffer obscurus, etc., in the genome sequence of puffer chrysanthemum (Takifugu flavidus, GenBank: GC...

Embodiment 2

[0111] This example discloses a polyclonal antibody, which is obtained by using the vitellogenin peptide (tfVWD) used for binding tetrodotoxin in Example 1 as an antigen, and preparing antiserum by immunizing mice.

[0112]The specific preparation process of the polyclonal antibody is as follows: 10 mice were immunized with the purified tfVWD protein obtained in Example 1 to prepare the polyclonal antibody. On the third day after the fourth immunization, a small amount of blood was collected from the eyes of the mice with a disposable needle, and the serum titer was detected by ELISA. Blood was collected 4 days after the last injection. Rat blood was collected in a 1.5mL sterile Eppendoff tube, marked with the tube number, and allowed to stand at room temperature for 2-3 hours. About 1 mL of blood can be collected from each mouse. After the blood coagulates, use a pipette tip to carefully peel off the blood clot along the tube wall, and place it in a refrigerator at 4°C over...

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Abstract

The present invention discloses a vitellogenin peptide fragment tfVWD used for binding to tetrodotoxin. The vitellogenin peptide fragment tfVWD comprises a polypeptide having an amino acid sequence asshown in SEQ ID NO: 1, or an active fragment of the polypeptide. The invention further discloses a nucleotide sequence encoding the vitellogenin peptide fragment tfVWD used for binding to the tetrodotoxin; as shown in SEQ ID NO:2, by taking the vitellogenin peptide fragment tfVWD as an antigen, and mice are immunized to prepare antiserum to obtain a polyclonal antibody. The invention further discloses a preparation method for the vitellogenin peptide fragment tfVWD, and the preparation method comprises the following steps: S1, the nucleotide sequence encoding the vitellogenin peptide fragmenttfVWD is obtained by cloning, and the nucleotide sequence is cloned to a recombinant expression vector; S2, induced expression and condition optimization are performed on the recombinant expression vector to obtain an expression product; and S3, the expression product is filtered, and is eluted through an affinity chromatography column, and elution peaks are collected to obtain recombinant protein. The vitellogenin peptide fragment tfVWD and the nucleotide sequence and polyclonal antibody thereof obtained by the invention have a good binding property with the tetrodotoxin (TTX), and lay a foundation for developing detoxification therapeutic drugs for the tetrodotoxin.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a vitellogenin peptide (tfVWD), its encoded nucleotide sequence and its polyclonal antibody. The invention also relates to a preparation method of the vitellogenin peptide tfVWD. Background technique [0002] Tetrodotoxin (TTX) is a small molecule alkaloid neurotoxin with the molecular formula C 11 h 17 o 8 N 3 , with a molecular weight of 319. It is an amino perhydroquinazoline compound and is one of the most toxic neurotoxins found in nature. Tetrodotoxin is a typical Na + Channel blocker, can interact with Na on the surface of muscle nerve cell membrane + Channel receptor specific binding, inhibiting Na in nerve cells + influx and block excitatory transmission between nerve and muscle. [0003] Poisoning incidents caused by tetrodotoxin often occur in China, Japan and some countries in Southeast Asia, mainly due to eating poisonous pufferfish by mistake. Scholars at home...

Claims

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Application Information

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IPC IPC(8): C07K14/46C07K16/18C07K16/06C07K1/36C07K1/34C07K1/22C12N15/12C12N15/70
CPCC07K14/461C07K16/18C07K16/065C12N15/70
Inventor 乔琨刘智禹黄文树蒋彩云陈贝许旻
Owner FISHERIES RES INST OF FUJIAN
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