Application of TREM-2 gene in identification of susceptibility of pigs to reproductive and respiratory syndrome virus

A blue ear virus and susceptibility technology, applied in the field of biomedicine, can solve the problems of PRRSV prevention and control, damage to the body's immune system, and vaccines without cross-protection, and achieve good clinical application value

Active Publication Date: 2016-12-21
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulty of prevention and control of PRRSV is mainly manifested in the following aspects: (1) macrophage and immunosuppressive diseases, PRRSV mainly infects pig alveolar macrophages (Porcinealveolar macrophages, PAMs), and PAMs are immune cells that destroy PAMs, thereby Destroy the body's immune system, thereby causing immunosuppression; (2) antigenic variability, currently PRRSV mutates rapidly, and the use of attenuated vaccines is one of the reasons for the virus to mutate. Recently, it has been reported in the literature that a new strain of PRRSV, NADC30, has appeared in the United States. In China, a new strain similar to that of the United States was also isolated, named NADC30-like, and another literature reported that a PRRSV-causing virus strain with a high degree of homology to the vaccine virus genome was isolated from a pig farm, and its

Method used

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  • Application of TREM-2 gene in identification of susceptibility of pigs to reproductive and respiratory syndrome virus
  • Application of TREM-2 gene in identification of susceptibility of pigs to reproductive and respiratory syndrome virus
  • Application of TREM-2 gene in identification of susceptibility of pigs to reproductive and respiratory syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 PAMs cell isolation and culture

[0037] 1. SPF level 6-8 week-old weaned piglets tested free of PRRSV, porcine circovirus type 2 (PCV-2), classical swine fever virus (CSFV) and other specific pathogens were bound, and the anterior vena cava was bled to death. The trachea was ligated, the chest was cut open, and the complete lungs were taken out together with the heart. The surface of the lungs was fully rinsed with sterile PBS to remove blood clots and dirt, and the heart was removed after cleaning. Pour into PBS buffer, gently pat and knead the lung surface for 5-10 minutes, and recover the bronchoalveolar lavage fluid. Repeat steps 2 to 3 times until a total of about 1000 mL of lavage fluid is recovered. The recovered alveolar lavage fluid was blown back and forth gently with a pipette to separate the cell clumps, the lavage fluid was centrifuged at 1000 rpm for 10 min at 4°C, and the supernatant was discarded. The cells were resuspended in RPMI 1640 medi...

Embodiment 2

[0039] Example 2 Study of TREM-2 gene regulating PRRSV replication in vitro

[0040] 1. Cultivate PAMs cells with RPMI 1640 medium containing 10% fetal bovine serum, add LPS to stimulate for 2 h, inoculate PRRSV at MOI=0.1, and continue to culture in 2% fetal bovine serum RPMI 1640 medium at 37°C for 24 hours h, cells were collected, and TREM-2 gene and PRRSV N protein were detected by qRT-PCR and Western-Blot respectively. The result is as figure 1 As shown in A, B, C, and D, after LPS stimulated PAMs cells, the expression of TREM-2 gene decreased, and PRRSV was inhibited at the same time.

[0041] 2. In addition, culture PAMs cells with RPMI 1640 medium containing 10% fetal bovine serum, inoculate PRRSV at MOI=0.1, continue to culture in RPMI 1640 medium containing 2% fetal bovine serum for 24 h at 37°C, and collect the cells. qRT-PCR and Western-blot detection were performed on TREM-2 gene respectively. The result is as figure 1 As shown in E and F, the expression of TR...

Embodiment 3

[0042] Example 3 Verification of the relationship between TREM-2 gene and ADAM17 and CD163

[0043] 1. Cultivate PAMs cells with RPMI 1640 medium containing 10% fetal bovine serum, add LPS to stimulate for 2 h, inoculate PRRSV at MOI=0.1, and continue to culture in 2% fetal bovine serum RPMI 1640 medium at 37°C for 24 hours h, cells were collected, and qRT-PCR was performed on CD163 and ADAM17 genes respectively.

[0044] 2. The result is as follows figure 2 As shown, after LPS stimulated PAMs cells, the expression of metalloproteinase ADAM17 was increased, while the expression of CD163 was inhibited.

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Abstract

The invention discloses an application of TREM-2 gene in identification of susceptibility of pigs to a reproductive and respiratory syndrome virus , and provides a group of TREM-2 expression level detection primer. The group of detection primers comprises an upstream primer TREM2-F represented by SEQ ID NO.1 and a downstream TREM2-R represented by SEQ ID NO.2. Researches show that the TREM-2 gene expression level in the pigs is related with PRRSV infection, and the TREM-2 can regulate the expression of CD163 in order to regulate duplication of the PRRSV; and the pigs can resist the PRRSV when the TREM-2 expression level is low, and the pigs are susceptible to the PRRSV when the TREM-2 expression level is high. The TREM-2 gene lays a solid foundation for identification of the susceptibility of pigs to the PRRSV and screening and cultivation of PRRSV-resistant pigs as a novel PRRSV susceptibility determination standard, and is of great significance to control the porcine reproductive and respiratory syndrome.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of TREM-2 gene in identifying susceptibility of pigs to PRRS virus. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV), first appeared in the United States in 1987, and then in reports from Europe. my country first isolated PRRSV in 1996 by Guo Baoqing and others, and named the isolated strain CH-1a, which confirmed the existence of the disease in my country. As early as 1992, the World Organization for Animal Health (Office International Des Epizooties, OIE) listed the disease as a Class B infectious disease. Currently, PRRS is one of the most important infectious diseases in the swine industry, causing huge economic losses to the swine industry worldwid...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124
Inventor 郭春和刘小红陈瑶生朱振邦
Owner SUN YAT SEN UNIV
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