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Application of MARCO in screening blue ear disease resistant pigs

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: 2019-01-15
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 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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  • Application of MARCO in screening blue ear disease resistant pigs
  • Application of MARCO in screening blue ear disease resistant pigs
  • Application of MARCO in screening blue ear disease resistant pigs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 PAMs cell isolation and culture

[0044] 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

[0046] Example 2 Research on the regulation of PRRSV replication by MARCO gene in vitro

[0047] 1. Cultivate PAMs cells with RPMI 1640 culture medium containing 10% fetal bovine serum, inoculate PRRSV at MOI=0.1, continue culturing in RPMI 1640 medium with 2% fetal bovine serum at 37°C, and collect cells at different time points. qRT-PCR and Western-blot detection were performed on the MARCO gene respectively. The result is as figure 2 , image 3 As shown, after PRRSV infected PAMs cells, the expression of MARCO was significantly reduced.

[0048] 2. Cultivate PAMs cells with RPMI 1640 medium containing 10% fetal bovine serum, inoculate PRRSV at different MOIs, continue culturing in RPMI 1640 medium containing 2% fetal bovine serum for 24 h at 37°C, collect cells, and MARCO gene was detected by qRT-PCR. The result is as Figure 4 As shown, after PRRSV infected PAMs cells, the expression of MARCO was significantly reduced.

Embodiment 3

[0049] Example 3 MARCO interference

[0050] 1. Send the designed MARCO 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). In two 6-well plates, culture PAMs cells to 60-70% density with RPMI 1640 medium containing 10% fetal bovine serum, respectively add siRNA (3 pairs of siRNA and 1 control) and Lipofectamine™ RNAiMAX with Opti-MEM ® Reduced Serum Medium was diluted, mixed at a ratio of 1:1 after dilution and incubated at room temperature for 5 min, and then added to PAMs cells replaced with fresh RPMI 1640 medium at 37°C, 5% CO 2 After culturing in an incubator for 6 h, the supernatant was discarded, washed once with PBS, and cultured in RPMI 1640 medium containing 10% fetal bovine serum for 48 h. Cells were collected to verify the effect of MARCO interference by qRT-PCR experiments. After another 6-well plate was cultured for 48 h, PRRSV wa...

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Abstract

The invention discloses the application of MARCO in screening blue ear disease resistant pigs. A study on that invention shows that the expression amount of MARCO in the pig body is closely related tothe infection of PRRSV, the expression of MARCO is inhibited after PRRSV is infected with PAMs, the interference of MARCO promotes PRRSV replication, and the overexpression of MARCO inhibits virus replication; when the expression of MARCO is abnormally high in vivo, the pigs are resistant to PRRSV, otherwise the pigs are susceptible to PRRSV. Therefore, MARCO can be used as a new criterion to judge the susceptibility or resistance of PRRSV. This study laid a solid foundation for identifying susceptibility to PRRSV and screening and breeding of PRRSV-resistant pigs, has good clinical application value for screening and breeding of PRRSV-resistant pigs, has important significance for prevention and control of blue-ear disease of pigs, and is also conducive to breeding excellent strain of pigs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to the application of MARCO in screening pigs for resistance 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). 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 economic losses to the pig industry. Highly pathogenic porcine reproductive and respi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/158
Inventor 郭春和张晓晓朱振邦俞飘董文娟贺胜王小瑛刘小红陈瑶生
Owner SUN YAT SEN UNIV
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