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Application of Def C protein in resisting dengue virus and expression method of Def C protein

An expression method and technology of dengue virus, which can be used in antiviral agents, peptide preparation methods, double-stranded DNA viruses, etc., can solve problems such as lethality, and achieve significant application value and guiding significance.

Pending Publication Date: 2022-01-04
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many dengue fever virus infections cause only mild illness, dengue fever virus can also cause acute flu-like illness, and sometimes develop into potentially fatal complications. However, there are no dengue fever vaccines and specific antiviral drugs on the market in the world at present. Supportive and symptomatic treatment measures, the principles of treatment are early detection, early diagnosis, early treatment, early mosquito prevention and isolation

Method used

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  • Application of Def C protein in resisting dengue virus and expression method of Def C protein
  • Application of Def C protein in resisting dengue virus and expression method of Def C protein
  • Application of Def C protein in resisting dengue virus and expression method of Def C protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The Aedes albopictus defensin family includes Def A, Def C and Def D, which have a wide range of antibacterial activities. The inventors have confirmed through experiments that Def C can be used as an important anti-dengue virus factor in Aedes albopictus.

[0045] This embodiment provides a method for obtaining the target recombinant Def C protein.

[0046] S1: Construction of recombinant protein vector;

[0047] Use Trizol (Invitrogen) to extract total RNA and synthesize cDNA from the midgut of Aedes albopictus, and then use the RNA extracted from the mosquito midgut as a template to perform reverse transcription; then according to the latest predicted Aedes albopictus Def C gene sequence in GenBank, A pair of full-length primers containing restriction restriction sites were designed using primer5.0 software. Wherein, the upstream primer sequence adopted is the nucleotide sequence shown in SEQ ID NO: 1, and the downstream primer sequence is the nucleotide sequence s...

Embodiment 2

[0065] In this example, on the basis of Example 1, the experiment is continued to confirm the toxicity of the recombinant Def C protein, and to verify that the recombinant Def C protein has the function of resisting dengue virus.

[0066] First, CCK-8 was used to detect the toxic effect of Aedes albopictus recombinant Def C protein on C6 / 36 cells and Vero cells, and the results are shown in the appendix Figure 4 shown. in, Figure 4 -a is the toxicity test result of recombinant Def C protein to C6 / 36 cells, Figure 4 -b is the toxicity test result of recombinant Def C protein to Vero cells.

[0067] Such as Figure 4 As shown, the Aedes albopictus Def C recombinant protein was diluted to 6 concentrations from 200 μg / mL, the recombinant DefC protein concentration varied from 0 μg / mL to 200 μg / mL (maximum concentration range), and the OD450 of each group of cells The values ​​were all similar, and the recombinant Def C protein was not toxic to C6 / 36 cells and Vero cells, an...

Embodiment 3

[0079] This example discloses the use of a polypeptide containing the Def C functional domain in the preparation of anti-dengue virus drugs. The amino acid sequence of the polypeptide is shown in SEQ ID NO:4.

[0080] In other preferred embodiments, the amino acid sequence shown in SEQ ID NO: 4 is substituted, deleted and / or increased by one or more amino acids and has the same function. It can be used to prepare anti-dengue virus drugs.

[0081]Based on the amino acid sequence of SEQ ID NO:4, synthesize the target peptide protein powder, fully dissolve it with 1mL LPBS and filter it with a 0.22μm filter to sterilize, aliquot and dilute a part to the working concentration for later use.

[0082] Next, the function of the polypeptide was tested.

[0083] First, CCK-8 was used to detect the toxic effect of the polypeptide on C6 / 36 cells and Vero cells, and the results are shown in the attached Figure 11 shown. in, Figure 11 -a is the result of the toxicity test of the poly...

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Abstract

The invention provides an application of Def C protein in resisting dengue virus and an expression method of the Def C protein, and particularly provides an application of recombinant Def C protein of aedes albopictus or polypeptide thereof in preparation of drugs for resisting dengue virus. Further, the nucleotide sequence of the recombinant Def C protein is SEQ ID NO: 3, or obtained by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence as shown in SEQ ID NO: 3 for expressing protein with the same function. The amino acid sequence of the polypeptide is SEQ ID NO: 4. According to the invention, the new application of the Def C protein or the polypeptide thereof in preparation of drugs for resisting dengue virus is disclosed for the first time, thereby providing breakthrough for development of the anti-dengue-virus drug and vaccine preparation field, and having important application value and guiding significance for clinical treatment and related theoretical researches.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, the present invention relates to the use of Def C protein for resisting dengue virus and its expression method. Background technique [0002] Dengue fever (DF) is an acute infectious disease caused by dengue virus (DENV) infection, which is mainly transmitted through the bite of Aedes aegypti and Aedes albopictus. Aedes albopictus has a wide distribution in Southeast Asia and my country, and is considered to be an important vector of dengue fever in my country. In recent years, the prevalence of dengue fever in my country and the number of infected people are on the rise, and the prevention and control situation is very severe. Although many dengue fever virus infections cause only mild illness, dengue fever virus can also cause acute flu-like illness, and sometimes develop into potentially fatal complications. However, there are no dengue fever vaccines and specific antiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P31/14C07K14/435C07K1/22C12N15/12C12N15/866
CPCA61K38/17A61P31/14C07K14/43563C12N15/86C12N2710/14043Y02A50/30
Inventor 梁韶晖刘文权陈晨
Owner WENZHOU MEDICAL UNIV
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