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A method for screening pigs resistant to PRRS based on the expression level of ext1

A PRRSV expression technology, applied in the field of biomedicine, can solve problems such as increased virulence, difficulty in preventing and controlling PRRSV, and damage to the immune system of the body

Active Publication Date: 2021-09-07
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 high-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, viremia can be detected in pigs for a long time , PRRSV persists in the body for up to 5 months; (6) Mixed infection, mixed infection of PRRSV and other diseases is common clinically at present, especially the combination of circovirus, Haemophilus parasuis, porcine pneumonia, etc. with PRRSV Mixed infection makes the prevention and control of PRRSV more difficult

Method used

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  • A method for screening pigs resistant to PRRS based on the expression level of ext1
  • A method for screening pigs resistant to PRRS based on the expression level of ext1
  • A method for screening pigs resistant to PRRS based on the expression level of ext1

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Experimental program
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Effect test

Embodiment 1

[0047] Example 1 Research on EXT1 gene regulating PRRSV replication in vitro

[0048] Marc-145 cells were cultured in DMEM medium containing 10% fetal bovine serum, and inoculated with three different strains of PRRSV (CHR6, TJM, PRRSV-EGFP) at MOI=0.1, in DMEM medium containing 2% fetal bovine serum Continue to culture at 37°C, collect cells at different time points, and perform Western-blot detection on the EXT1 gene. The results are as follows: figure 1 As shown, after PRRSV infected Marc-145 cells, the expression of EXT1 protein was significantly increased. qRT-PCR detection of EXT1 gene, the results are as follows figure 2 , 3 As shown in , 4, after three different strains of PRRSV (CHR6, TJM, PRRSV-EGFP) infected Marc-145 cells, the expression of EXT1 mRNA was significantly increased.

Embodiment 2

[0049] Embodiment 2 EXT1 interference

[0050] 1. Send the designed EXT1 siRNA and 1 pair of Negative Control (control) to Thermo Fisher Scientific for synthesis, and use Lipofectamine™ RNAiMAX transfection reagent (purchased from Thermo Fisher Scientific). Culture Marc-145 cells to 60-70% density in 2 6-well plates with DMEM medium containing 10% fetal bovine serum, and respectively mix siRNA (3 pairs of siRNA and 1 control) and Lipofectamine™ RNAiMAX with Opti-MEM ®Reduced Serum Medium diluted, mixed at a ratio of 1:1 after dilution and incubated at room temperature for 5 minutes, then added to Marc-145 cells replaced with fresh DMEM medium and incubated in a 37°C, 5% CO2 incubator for 6 hours, discarded After washing once with PBS, the cells were cultured in DMEM containing 10% fetal bovine serum for 48 h, and the cells were collected to verify the interference effect of EXT1 by qRT-PCR. After another 6-well plate was cultured for 48 h, PRRSV was added to the culture mediu...

Embodiment 3

[0054] Example 3 EXT1 overexpression

[0055] 1. Construct pcDNA3.1-EXT1 overexpression vector, culture Marc-145 cells to 60-70% density with DMEM medium containing 10% fetal bovine serum in two 6-well plates, use Lipofectamine™ 2000 (purchased from Thermo Fisher Scientific) respectively transferred pcDNA3.1-EXT1 and pcDNA3.1 empty vectors into Marc-145 cells, cultured in a 37°C, 5% CO2 incubator for 6 h, discarded the supernatant, washed once with PBS, and washed with 10% The DMEM medium of fetal bovine serum was cultured for 24 h, and the cells were collected to detect the effect of overexpression. After another 6-well plate was cultured for 24 hours, PRRSV was added to the culture medium, and then cultured for 36 and 48 hours, and the cells were collected to detect the effect of EXT1 overexpression on PRRSV replication.

[0056] 2. The result is as follows Figure 9 As shown, after EXT1 overexpression, EXT1 mRNA was significantly increased. EXT1 is a heparan sulfate HS s...

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Abstract

The invention discloses the application of EXT1 in screening pigs resistant to PRRS and a method for screening pigs resistant to PRRS based on the expression level of EXT1. The research of the present invention shows that the expression level of EXT1 in pigs is closely related to PRRSV infection. After PRRSV infects Marc-145 cells, EXT1 mNRA and protein levels increase, and EXT1 interference promotes PRRSV replication, and EXT1 overexpression inhibits virus replication; when EXT1 expression in vivo is higher When , pigs are resistant to PRRSV, and vice versa. Therefore, EXT1 can be used as a new type of criterion for judging PRRSV susceptibility or resistance, which lays a solid foundation for the identification of pigs' susceptibility to PRRSV and the screening and breeding of PRRSV-resistant pigs. The screening and breeding of PRRSV-resistant pigs It has good clinical application value, is of great significance to the prevention and control of porcine PRRS, and is also conducive to the cultivation of excellent strains of pigs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to the application of EXT1 in screening pigs resistant to PRRS and a method for screening pigs resistant to PRRS based on the expression level of EXT1. 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). As early as 1992, the World Organization for Animal Health (OfficeInternational Des Epizooties, OIE) listed the disease as a Class B infectious disease. Currently, PRRS is one of the most important infectious diseases in the pig industry, which is distributed in almost all pig farming countries and causes huge economic losses to the pig industry worldwide. In 2006, porcine high fever syndrome (PHFS) broke out in most provinces of my country and Vietnam, causing huge eco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11A61K38/17A61K45/00A61P31/04
CPCA61K38/1709A61K45/00A61P31/04C12Q1/6888C12Q2600/124C12Q2600/158
Inventor 郭春和董文娟朱振邦张晓晓俞飘贺胜王小瑛刘小红陈瑶生
Owner SUN YAT SEN UNIV
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