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Application of calpain protein-1 in resisting porcine epidemic diarrhea virus infection

A calpain, gene technology, applied in the direction of antiviral agents, applications, viruses/phages, etc., to achieve high activity and significant anti-PEDV virus effect

Inactive Publication Date: 2021-11-09
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of immune strategies that can provide effective intestinal mucosal protection for PEDV infection, preventing and controlling the occurrence of PEDV has become a research problem in the world

Method used

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  • Application of calpain protein-1 in resisting porcine epidemic diarrhea virus infection
  • Application of calpain protein-1 in resisting porcine epidemic diarrhea virus infection
  • Application of calpain protein-1 in resisting porcine epidemic diarrhea virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Extraction of pig small intestine mucus and evaluation of antiviral effect

[0035] 1.1 Preparation of porcine small intestine mucus

[0036] 260-day-old fattening pigs were fasted for 3 hours before slaughter, and were euthanized by intravenous injection of sodium pentobarbital (100 mg / kg). Pig death is confirmed when the pig's corneal reflex disappears, breathing stops, and the heart stops beating. Subsequently, immediately dissected and sampled. The collection of small intestine mucus was carried out according to the previous research report, and appropriate adjustments were made. The specific steps are as follows: First, cut the small intestine about 10 cm long, turn it everted and put it on the glass column, stretch it fully and lay it flat Place in a pre-cooled glass petri dish on ice; then, add 5ml of pre-cooled PBS (containing 1% protease inhibitor cocktail), and scrape the mucus gently after one minute, taking care not to damage the intestinal mucos...

Embodiment 2

[0050] Example 2 Identification of Antiviral Activity of Each Component Protein in Porcine Small Intestinal Mucus

[0051] 2.1 Separation of protein components in porcine small intestinal mucus

[0052] The total porcine small intestinal mucus protein (10mg / mL) prepared in Example 1 was added to an Amicon Ultra 100kDa ultrafiltration tube, and the mucus total protein was divided into high molecular weight proteins (HMWPs) and low molecular weight proteins (LMWPs) by centrifugation. HMWPs and LMWPs were processed as follows: 1) Use Protein A / G affinity column to remove immunoglobulins in HMWPs, so as to exclude the interference of immunoglobulins on subsequent antiviral effect detection; 2) Separate LMWPs by non-denaturing gel separation Divide into 4 protein fractions (I-IV), and use electroelution method to purify and collect ( figure 2 A).

[0053] 2.2 Detection of antiviral effects of protein components in porcine small intestinal mucus

[0054] The antiviral effect of ...

Embodiment 3

[0055] Example 3 Identification of proteins with antiviral activity in porcine small intestinal mucus LMWPs fraction III

[0056] Using molecular sieves (SEC, Zenix SEC-150) to analyze and purify the protein in LMWPs fraction III, the main protein absorption peak was detected at 8.313s, accounting for about 74.79% of the total area of ​​protein fraction III ( image 3 A). After the main peak protein was collected, the molecular weight of the protein was found to be about 70kD after SDS-PAGE electrophoresis, and the antiviral activity of the collected protein was further verified. The purified protein fraction III was mixed with an equal volume of PEDV (10 4 PFU) were incubated for 1 h, and the mixture was inoculated into Vero E6 cells. After culturing for 1 h, the mixed solution was washed away, and the antiviral activity of protein component III was determined by plaque inhibition test. image 3 The result of C shows that the collected protein can significantly inhibit the...

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Abstract

The invention discloses Calpain-1 and application thereof in inhibiting porcine epidemic diarrhea virus infection, and belongs to the technical field of biological engineering. On the basis of finding that pig small intestine mucus total protein can inhibit PEDV infection, the proteinase-1 capable of inhibiting PEDV invasion into host cells is obtained through identification and purification from pig small intestine mucus, the nucleotide sequence of the protein coding gene is as shown in SEQ ID No.1 in a sequence table, and the amino acid sequence of the protein coding gene is as shown in SEQ ID No.2. The porcine calpain protein-1 further obtained through eukaryotic expression also has a remarkable antiviral function, and the porcine calpain protein-1 is mainly used for inhibiting PEDV from invading host cells through enzymolysis of PEDV spike protein. The Calpain-1 disclosed by the invention can also be applied to the preparation of PEDV infection resisting medicines or antiviral feed additives.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to the application of calpain-1, a mucus protein in the small intestine of pigs. The calpain-1 obtained by screening, separating and identifying the mucus in the small intestine of pigs can significantly inhibit PEDV infection, and the anti-virus mechanism of calpain-1 lies in its It can enzymatically hydrolyze PEDV S protein and inhibit the virus from invading host cells. Background technique [0002] The intestinal mucosa constitutes the largest interface between the body and the outside world, and is an important site for many pathogenic microorganisms to invade and establish infection. The mucus layer covering the surface of intestinal epithelial cells plays an important role in maintaining the structural and functional integrity of porcine small intestinal mucosa. Mucus is mainly secreted by intestinal secretory cells, such as goblet cells and Paneth cells, and its composition is c...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N15/57C12N15/85C12N5/10A61K38/48A61P31/14A23K20/189A23K50/30
CPCC12N9/6472C12N15/85C12N5/0686A61P31/14A23K20/189A23K50/30C12Y304/22017C12N2800/107C12N2510/02A61K38/00Y02A50/30
Inventor 李昱辰杨倩王秀羽张恩
Owner NANJING AGRICULTURAL UNIVERSITY
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