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Blocking agent capable of inhibiting porcine reproductive and respiratory syndrome virus infection

A technology for respiratory syndrome and virus infection, applied in the field of biology, can solve problems such as weakened immune protection, and achieve the effect of expanding the scope of research and reducing the number of infected cells

Active Publication Date: 2019-05-03
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the emergence of NADC30-like PRRSV, new mutant strains composed of this strain and HP-PRRSV have been recombined. The mutant strains not only change in genome, but also in virulence and induced immune response of the body Changes have taken place in all aspects, resulting in the weakening of the immune protection of existing vaccines, resulting in continuous outbreaks and epidemics of PRRS. Therefore, it is urgent to find drugs or effective vaccines that have therapeutic effects on both HP-PRRSV and LP-PRRSV

Method used

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  • Blocking agent capable of inhibiting porcine reproductive and respiratory syndrome virus infection
  • Blocking agent capable of inhibiting porcine reproductive and respiratory syndrome virus infection
  • Blocking agent capable of inhibiting porcine reproductive and respiratory syndrome virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Determination of SRCAP proteins interacting with PRRSV nonstructural protein NSP4

[0053] SRCAP is a transcriptional activator of the Notch signaling pathway, which can regulate the transcription of Notch signaling pathway effector genes, and also belongs to the ATP-dependent chromatin remodeling enzyme of the IN080 family. We previously connected the PRRSV nonstructural protein nsp4 gene to the eukaryotic expression vector , expressed in 293T cells, the cell protein that may interact with NSP4 was collected by GFP-Trap agarose beads, and the cell protein SRCAP was found to interact with NSP4 through mass spectrometry analysis and Western blot verification (see figure 1 A), and the co-localization phenomenon of cellular protein SRCAP and NSP4 was also observed by confocal microscope (see figure 1 B).

Embodiment 2

[0054] Example 2: Identification of SRCAP involved in PRRSV infection in Marc-145 cells

[0055] In order to further study the involvement of SRCAP in PRRSV infection, according to the results of mass spectrometry, siRNA of SRCAP was designed, and after its expression was reduced, the effect on HP-PRRSV and LP-PRRSV infection was analyzed.

[0056] According to the SRCAP gene sequence published by NCBI (GenBank accession number: NM_006662.3), the 1285th, 1694th and 2108th siRNA gene silencing site sequences of SRCAP (Monkey) and the 1736th siRNA gene sequence of SRCAP (Swine) were designed using premier6.0 primer design software , 7756 siRNA gene silencing site sequences and negative control sequences (see Table 1). After the sequence was designed, it was sent to Shanghai Gemma Pharmaceutical Technology Co., Ltd. for synthesis. The synthesized sequence was diluted with DEPC water to 1 μM, dispensed into RNase-free 1.5mL centrifuge tubes, and stored at -20°C.

[0057] Select M...

Embodiment 3

[0062] Example 3: Knockdown of SRCAP on PAM and its effect on HP-PRRSV and LP-PRRSV infection

[0063] In order to further verify the results, primary porcine lung macrophages (PAM) were used for verification. The 50-day-old SPF piglets were bled, their lungs were aseptically removed after the trachea was ligated, and the outer surface was washed with autoclaved PBS (1640 medium with 1 / 25 volume of PBS and 5× double antibody), and the pH7 .2 Infuse 30.0ml-50ml of PBS into the lungs from the trachea, gently pat the lung surface, recover the lavage fluid after 1-2 minutes, and repeat this process until the lavage fluid is clear. Gently blow the recovered bronchoalveolar lavage fluid with a straw to break up the cell clumps, filter through a single-layer sterile 100-mesh stainless steel sieve, collect all the lavage fluid, centrifuge at 1500r / min for 5-10min, and collect the precipitate. Wash twice with PBS containing 5× double antibody, each time gently mix and centrifuge. The...

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Abstract

The invention discloses a blocking agent capable of inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection. The invention discovers a new PRRSV cell target protein, namely Snf2-related CBP activator protein (SRCAP), and finds out inhibitory targets of the receptor. PRRSV infection can be significantly reduced by interfering SRCAP genes or using a Notch signaling pathway inhibitor, namely a gamma-secretase inhibitor, in which the SRCAP protein plays a key role at the inhibitory targets; and thus, the inhibitory targets and the gamma-secretase inhibitor can be developedinto drugs used for preventing and treating PRRSV infection, thereby providing brand new ideas for PRRSV research as well as prevention and treatment. Therefore, scope of research is expanded; and thus, the blocking agent is of great significance to practical production.

Description

technical field [0001] The invention relates to the technical field of biology, in particular to a blocking agent for inhibiting porcine reproductive and respiratory syndrome virus infection. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). The disease is mainly characterized by reproductive impairment in sows and dyspnea in piglets, causing huge economic losses to the swine industry. PRRSV has a high degree of variability. The virus was first isolated in my country in 1996, and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) appeared 10 years later. In 2013, NADC30-like PRRSV appeared. Since the emergence of NADC30-like PRRSV, new mutant strains composed of this strain and HP-PRRSV have been recombined. The mutant strains ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K38/16A61K31/713A61P31/14C12N15/113
Inventor 肖一红丁国飞蔡鑫娜刘思当
Owner SHANDONG AGRICULTURAL UNIVERSITY
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