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Application of protein CbAE with broad-spectrum antiviral function

A protein and virus technology, applied in antiviral agents, peptide/protein components, medical preparations containing active ingredients, etc., can solve problems such as large side effects, nucleoside drug resistance, and virus mutations

Active Publication Date: 2020-08-25
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current treatment methods for viruses are mainly treated with antiviral drugs (such as interferon and nucleoside drugs). Interferon treatment has relatively large side effects, and nucleoside drugs are more prone to drug resistance; Viral diseases, generally treated symptomatically
Because viruses are more prone to mutations, there are currently no specific vaccines for most viruses

Method used

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  • Application of protein CbAE with broad-spectrum antiviral function
  • Application of protein CbAE with broad-spectrum antiviral function
  • Application of protein CbAE with broad-spectrum antiviral function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Embodiment 1, the acquisition of fusion protein CbAE-1

[0134] 1. Construction of recombinant plasmid pET-28a(+)-CbAE-1

[0135] The small DNA fragment between the restriction endonuclease EcoRI and the HindIII recognition sequence of the vector pET-28a (+) is replaced by the double sequence shown in the 1st to 1356th position of SEQ ID NO: 1 from the 5' end. Stranded DNA molecules were obtained to obtain the recombinant plasmid pET-28a(+)-CbAE-1.

[0136] In the recombinant plasmid pET-28a(+)-CbAE-1, the DNA molecule shown in the 1st to 1356th position from the 5' end of SEQ ID NO: 1 and the His-tag tag on the vector backbone (consisting of 6 histidines The coding sequence of residue composition) is fused to form the fusion gene CbAE-1 shown in SEQ ID NO: 3, and express the fusion protein CbAE-1 with His-tag tag shown in SEQ ID NO: 4.

[0137] 2. Expression and purification of fusion protein CbAE-1

[0138] Bacterial lysate: 50 mM Tris-HCl buffer (pH8.0) containing...

Embodiment 2

[0161] Embodiment 2, the acquisition of fusion protein CbAE-2

[0162] 1. Construction of recombinant plasmid pET-28a(+)-CbAE-2

[0163] The DNA fragment between the restriction endonuclease EcoRI and the HindIII recognition sequence of the vector pET-28a (+) is replaced with the double sequence shown in the 1st to 1332nd position of SEQ ID NO: 5 from the 5' end. Stranded DNA molecules were obtained to obtain the recombinant plasmid pET-28a(+)-CbAE-2.

[0164] In the recombinant plasmid pET-28a(+)-CbAE-2, the DNA molecule shown in position 1 to 1332 from the 5' end of SEQ ID NO: 5 and the His-tag tag on the vector backbone (composed of 6 histidines Residue composition) coding sequence is fused to form the fusion gene CbAE-2 shown in SEQ ID NO: 7, expressing the fusion protein CbAE-2 with His-tag tag shown in SEQ ID NO: 8.

[0165] 2. Expression, purification, renaturation and endotoxin removal of fusion protein CbAE-2

[0166] According to the method of step 2 and step 3 of...

Embodiment 3

[0168] Embodiment 3, the inhibitory action of fusion protein CbAE-1 and fusion protein CbAE-2 to novel coronavirus pseudovirus

[0169]The mixed reagent is made by mixing 1vial Luciferase Assay Substrate (lyophilized) and 10mL Luciferase Assay Buffer. 1 Vial Luciferase Assay Substrate (lyophilized) and Luciferase Assay Buffer are components in Luciferase Assay System (Promega, E1500).

[0170] 1×lysis buffer is prepared by mixing 1 volume part of 5×lysis buffer (product of Promega Company, item number is E1500) and 4 volume parts of water.

[0171] 1. Inhibitory effect of fusion protein CbAE-1 on novel coronavirus pseudovirus

[0172] 1. Digest HEK-293T cells stably expressing ACE2 protein with 0.25% trypsin without EDTA, then re-infect with DMEM medium containing 10% (v / v) FBS, 100U / mL streptomycin and 100U / mL penicillin suspended to obtain a concentration of 1.3×10 6 cells / mL of cell suspension.

[0173] 2. Take a 96-well plate, add 100 μL of cell suspension to each well...

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Abstract

The invention discloses an application of a protein CbAE with a broad-spectrum antiviral function. The protein CbAE sequentially comprises a segment 1-segment 12, wherein the amino acid sequences of the segment 1 to the segment 12 are respectively as shown in the 72nd to 82nd positions, the 98th to 107th positions, the 109th to 114th positions, the 184th to 190th positions, the 218th to 242nd positions, the 232nd to 238th positions, the 273th to 283th positions, the 315 to 319 th positions, the 339 to 344 th positions, the 389 to 393 th positions, the 419 to 423 th positions, and the 425 to 429 th positions from the N end of SEQ ID NO: 2. Experiments prove that the fusion protein CbAE-1 and the fusion protein CbAE-2 can significantly inhibit novel coronavirus pseudo, novel coronavirus, HIVpseudo, DENV-2, ZIKV and HSV-1. The method has an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of a protein CbAE with broad-spectrum antiviral function. Background technique [0002] Coronaviruses are a type of enveloped, single-stranded positive-sense RNA virus, named for the crown-like shape of virus particles under an electron microscope. The host range of coronaviruses mainly includes birds and mammals. So far, seven coronaviruses that can infect humans have been identified, including SARS virus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and the new The emergence of coronavirus (SARS-CoV-2) has posed a great threat to the world's economic health. The novel coronavirus belongs to the family Coronaviridae and belongs to the genus Coronavirus. After infecting humans, it can cause symptoms of varying degrees, with fever, fatigue, and dry cough as the main manifestations. In severe cases, pneumonia, acute respiratory distres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K38/08A61P31/14A61P31/18A61P31/22C12R1/19
CPCC07K7/06C12N15/70A61P31/14A61P31/18A61P31/22C07K2319/00A61K38/00Y02A50/30
Inventor 程功余茜童良琴张礼铭肖小平朱毅斌
Owner TSINGHUA UNIV
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