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Application of hpse gene in screening pigs resistant to PRRS

A technology for PRRSV and PRRSV, which is applied in the field of biomedicine, can solve the problems of PRRSV prevention and control, the lack of cross-protection of vaccines, and damage to the immune system of the body, and achieve good clinical application value.

Active Publication Date: 2019-11-26
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
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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 virulence was enhanced. Analysis may be possible (3) The vaccine has no cross-protection, and PRRSV vaccines on the market have almost no cross-protection, and there is no cross-protection between different strains; (4) Antibody dependence is enhanced, and the PRRSV Infection will stimulate the body to produce antibodies, but low-titer antibodies not only cannot neutralize the virus, but can promote the proliferation of the virus; (5) The virus is persistently infected. After PRRSV infection, the virus blood can be detected in pigs for a long time syndrome, PRRSV lasts up to 5 months in the body; (6) mixed infection, mixed infection of PRRSV and other diseases is common in clinical practice, especially circovirus, Haemophilus parasuis, porcine pneumonia, etc. and PRRSV The mixed infection of PRRSV makes the prevention and control of PRRSV even more difficult

Method used

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  • Application of hpse gene in screening pigs resistant to PRRS
  • Application of hpse gene in screening pigs resistant to PRRS
  • Application of hpse gene in screening pigs resistant to PRRS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 PRRSV infection inhibits the expression of heparan sulfate HS

[0041] 1. Cultivate Marc-145 cells with DMEM medium containing 10% fetal bovine serum to a confluence of 60-70%, wash once with PBS, replace with DMEM medium containing 2% fetal bovine serum, and set MOI=0.1 Add PRRSV at 37°C, 5% CO 2 Cultured in a humidified incubator for 0, 24, and 36 h. Wash 3 times with PBS, fix with 4% paraformaldehyde for 10 min, wash with PBS for 10 min, perforate with 10% Triton-100 for 15 min, wash with PBS for 10 min, then block with 1% BSA diluted in PBS for 30 min, add anti-HSPG2 protein for one Anti-mouse (diluted 1:200) was incubated overnight at 4°C, anti-mouse secondary antibody (diluted 1:1000) was used for 1 h, nuclei were stained with DAPI dye for 5 min, and washed with PBS for 10 min. First check whether the heparan sulfate HS is stained under an ordinary fluorescent inverted microscope, and then observe under a confocal microscope.

[0042] 2. The result is...

Embodiment 2

[0043] Example 2 PRRSV infection activates NF-κB signaling pathway

[0044] 1. Culture Marc-145 and PAMs cells with DMEM and RPMI 1640 medium containing 10% fetal bovine serum respectively to a confluence of 60-70%, wash once with PBS, and replace with DMEM and RPMI 1640 containing 2% fetal bovine serum Culture medium, and add PRRSV at a ratio of MOI=0.1 at 37°C, 5% CO 2 Cultured in a humidified incubator for 0, 6, 12, 24, and 36 h. The p65 subunit of the NF-κB signaling pathway was detected by confocal and qRT-PCR according to the above method, and the cells were collected for Western Blot detection of the IκBα subunit of the NF-κB signaling pathway.

[0045] 2. The result is as follows figure 2 with 3 As shown, after PRRSV infects PAMs cells, the p65 subunit of NF-κB signaling pathway enters the nucleus ( figure 2 A), increasing the expression of p65 subunit of NF-κB signaling pathway ( figure 2 B). After PRRSV infected PAMs and Marc-145 cells, the phosphorylation of...

Embodiment 3

[0047] Example 3 PRRSV infection promotes HPSE gene expression

[0048] 1. Culture Marc-145 and PAMs cells with DMEM and RPMI 1640 medium containing 10% fetal bovine serum respectively to a confluence of 60-70%, wash once with PBS, and replace with DMEM and RPMI 1640 containing 2% fetal bovine serum Culture medium, and add PRRSV at a ratio of MOI=0.1 at 37°C, 5% CO 2 Cultured in a humidified incubator for 0, 6, 12, 24, and 36 h. Cells were collected for Western Blot detection of heparanase encoded by HPSE gene.

[0049] 2. The result is as follows Figure 4 As shown, PRRSV infects PAMs ( Figure 4 A) and Marc-145 ( Figure 4 B) after cells, the expression level of heparanase Heparanase encoded by HPSE gene is gradually increased. That is, PRRSV promotes the expression of HPSE gene after infecting PAMs and Marc-145 cells.

[0050] The expression of HPSE gene increased, and the activity of heparanase increased, which in turn cut HS, inhibited PRRSV particles from adhering ...

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Abstract

The invention discloses application of an HPSE gene in screening of pigs with resistance to the reproductive and respiratory syndrome, and provides a group of HPSE gene expressive quantity detection primers. The primers comprise an upstream primer HPSE-F and a downstream primer HPSE-R, and respectively have sequences as shown in the SEQ ID NO.1-2. Researches indicate that the HPSE gene expressive quantity in a big body is closely related to PRRSV infection, the HPSE gene coded heparanase is used for regulating PRRSV release and infection efficiency by regulating the expressive quantity of heparan sulfate on a cytomembrane; when the HPSE expressive quantity is low, a pig has resistance to PRRSV, and is easily affected otherwise. HPSE gene can be used as a novel standard for determining the susceptibility or resistance to PRRSV, and lays a solid foundation for screening and culture of PRRSV-resistance pigs, and has an important meaning for prevention and control of 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 HPSE gene in screening pigs resistant to PRRS. 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 worldwide. In 2005, PRRS ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/701
Inventor 郭春和刘小红陈瑶生朱振邦
Owner SUN YAT SEN UNIV
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