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Human alpha-defensin 5 antiviral active mutant polypeptide and preparation method and application

An antiviral activity and mutant technology, which is applied in the preparation methods of peptides, antiviral agents, botanical equipment and methods, etc., can solve the problems such as the decline of antibacterial and antiviral activities of defensins, and achieve the improvement of antiviral ability. Effect

Active Publication Date: 2010-05-12
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have confirmed that when the positively charged arginine (basic amino acid) in the defensin molecule is replaced with a neutral amino acid or an acidic amino acid, the antibacterial and antiviral activities of the defensin will be significantly reduced

Method used

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  • Human alpha-defensin 5 antiviral active mutant polypeptide and preparation method and application
  • Human alpha-defensin 5 antiviral active mutant polypeptide and preparation method and application
  • Human alpha-defensin 5 antiviral active mutant polypeptide and preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1HD5

[0071] The solid-phase chemical method synthesis of embodiment 1HD5 mutant polypeptide:

[0072] 1. According to the amino acid sequence of the HD5 mutant, use a fully automatic peptide synthesizer (433A, Applied Biosystem) to entrust Shanghai Sangon Bioengineering Company to synthesize the HD5 mutant polypeptide.

[0073] 2. Purify the synthesized HD5 mutant polypeptide by HPLC reverse phase C18 column chromatography and desalting.

[0074] 3. Use the same method to synthesize and purify the maternal HD5 polypeptide reference substance.

[0075] 4. For the purified HD5 mutant polypeptide, use high pressure liquid chromatography (HPLC) method for purity identification, apply matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) to determine its molecular weight, and determine its molecular weight by isoelectric focusing electrophoresis. The isoelectric point was determined by an automatic amino acid sequencer.

[0076] 5. The results of HPL...

Embodiment 2

[0077] Example 2 Expression and Purification of Recombinant HD5 Mutant Polypeptides in Pichia pastoris

[0078] 1. Synthesize the full-length gene encoding the HD5 mutant. Entrusted Shanghai Sangon Bioengineering Company to synthesize the full-length gene encoding the HD5 mutant, the gene sequence is as follows:

[0079] GCC ACC TGC TAT TGC CGA ACC GGC CGT TGT GCT ACC CGT GAG TCC CTC TCCGGG GTG TGT ATC AGT GGC CGC CTC TAC AGA CTC TGC TGT CGC

[0080] Note: The amino acid encoded by the 3 bases in the shaded part is the mutated arginine, and the 3 bases in the box encode the stop codon.

[0081] 2. Construction of recombinant HD5 mutant yeast expression vector

[0082] (1) PCR primer design: In order to ensure that there is no redundant amino acid at the N-terminal of the recombinant HD5 mutant expressed in yeast, the gene sequence encoding the HD5 mutant was directly inserted into the STE of the yeast expression vector pPIC9K using Red / ET recombination technology 13 aft...

Embodiment 3

[0091] Cytotoxicity assay of embodiment 3HD5 mutant polypeptide

[0092] Inoculate vero cells (African green monkey kidney cells) in a 96-well plate with a seeding density of 2000 cells / well and a seeding volume of 100 μl. The vero cells were cultured in 1640 culture medium containing 10% fetal bovine serum for 24 hours until the cells were completely attached. The culture medium was discarded, and the culture medium containing different concentrations of HD5 mutant polypeptides was replaced to continue the culture. Each concentration was repeated for 5 wells, and a maternal HD5 polypeptide control group and a blank control group were set at the same time. After culturing for 72 hours, add 10 μl of CCK-8 reaction solution to each well, continue culturing at 37°C for 4 hours, discard the supernatant gently, shake and mix well, and measure the absorbance value (A value) at 490 nm wavelength with a microplate reader. Cell survival rate (%)=A value of the drug group / A value of the b...

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Abstract

The invention relates to human alpha-defensin 5 (HD5) antiviral active mutant polypeptide and preparation method and application. The 21th glutamic acid in HD5 of the amino acid sequence of the mutant polypeptide can be replaced into arginine (E21R). The preparation method of the mutant polypeptide has two preparation methods, namely a solid-phase chemical synthesis method and a gene engineering preparation method. The HD5 mutant polypeptide of the invention has efficient antiviral activity under the conditions that serum exits or absents, and compared with parent HD5 polypeptide, the antiviral function of the invention is obviously improved. The HD5 mutant polypeptide of the invention is used for preventing viral infection, such as HSV, HPV, HIV and the like.

Description

technical field [0001] The invention belongs to the technical fields of protein engineering and preparation of antiviral drugs, and in particular relates to a mutant polypeptide with antiviral activity of human α-defensin 5 and its preparation method and application. Background technique [0002] Viral infectious diseases seriously endanger human health, and the development of highly effective and specific antiviral drugs has important practical significance. [0003] Defensin is an endogenous cationic peptide with a molecular weight of 3-6kD discovered in the process of studying the defense and resistance of pathogenic microorganisms and infections in mammals and insects. The reason why it is called a cationic peptide is that the mature defensin molecule is rich in arginine (a basic amino acid), so in most cases the defensin molecule has a strong positive charge. People's initial understanding of defensins was based on their rapid and potent bactericidal activity, but late...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/04C12N15/12C12N15/63C12P21/02A61K38/17A61P31/18A61P31/20A61P31/22C12R1/19C12R1/84
CPCC07K14/4723A61K38/1709A61P31/04A61P31/10A61P31/18A61P31/20A61P31/22
Inventor 王军平陈芳王艾平申明强王崧粟永萍程天民
Owner ARMY MEDICAL UNIV
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