Porcine reproductive and respiratory syndrome mutant virus as well as construction method and application thereof

A technology for respiratory syndrome and mutant viruses, applied in applications, viruses, viral peptides, etc., can solve the problem of strong virulence

Pending Publication Date: 2021-11-02
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Vaccines are considered to be an important tool and means for the prevention, control and purification of PRRSV. However, there are various problems in the commercialized PRRSV vaccines. Among them, the long-term use of live attenuated vaccines has potential risks such as virulence reversion. Key factor

Method used

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  • Porcine reproductive and respiratory syndrome mutant virus as well as construction method and application thereof
  • Porcine reproductive and respiratory syndrome mutant virus as well as construction method and application thereof
  • Porcine reproductive and respiratory syndrome mutant virus as well as construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0035] Construction of embodiment 1 mutant virus RvJXwn-nsp1α-G90A full-length cDNA clone plasmid

[0036] Referring to the complete genome sequence of PRRSV JXwn06 (GenBank accession number EF641008), primers were designed for the construction of the full-length cDNA clone of PRRSV mutant strain RvJXwn-nsp1α-G90A (see Table 1 below). The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. Use nuclease-free water to prepare the concentration for use.

[0037] Table 1 Primers for construction of RvJXwn-nsp1α-G90A full-length cDNA clone

[0038]

[0039] Note: The sequence in bold italic is the recognition sequence of restriction endonuclease

[0040] The construction strategy of the mutant strain RvJXwn-nsp1α-G90A full-length cDNA cloning plasmid is shown in the appendix figure 1 . Use high-fidelity DNA polymerase KOD-Fx-Neo (Toyobo, Shanghai, China) to amplify the target fragment, and construct a 50 μL reaction system according to the product manual. The amp...

Embodiment 2

[0043] Example 2 Rescue of mutant virus RvJXwn-nsp1α-G90A

[0044] Inoculate well-growing MARC-145 cells in 6-well cell plates at a ratio of 1:3, and place at 37°C with 5% CO 2 Culture in an incubator until the cell density reaches 80%-90% for transfection. According to the instructions of Lipofectamine LTX (Invitrogen, CA, USA) transfection reagent, 200 μL of Opti-MEM (Invitrogen, CA, USA) was added to a sterile 1.5ml EP tube, and then 2.5 μg of full-length infectious cloning plasmid and PLUS Reagents 2.5μL, mix thoroughly, and let stand at room temperature for 5min. Then add Lipofectamine LTX 7.5μL, mix thoroughly, and let stand at room temperature for 20min. Then the mixture was slowly added dropwise to the cell culture plate, and 24 hours after the transfection, the DMEM medium containing 10% FBS (fetal bovine serum) was replaced with fresh, and the culture was continued for 3d-5d. After the typical PRRSV cell lesions appear, that is, the cells are initially focally rou...

Embodiment 3

[0045] Identification of embodiment 3 mutant virus RvJXwn-nsp1α-G90A

[0046] RT-PCR Identification of Rescued Viruses

[0047] Take 250 μL of the rescued F3 virus solution and add it to a 1.5mL centrifuge tube, then add 1mL Trizol to mix, and let stand at room temperature for 10min; add 200μL of chloroform, shake vigorously for 15s, and let stand at room temperature for 10min; centrifuge at 4°C, 12000g for 15min , carefully pipette 450 μL of the supernatant into a new 1.5 mL centrifuge tube; add 650 μL of isopropanol, stand at -20°C for 15 minutes to overnight, centrifuge at 12,000 g for 15 minutes at 4°C, discard the supernatant; add 750 μL of 75% ethanol to wash the precipitate, Centrifuge at 4°C and 7500g for 6 minutes, discard the supernatant; dry the pellet at room temperature for 10 minutes, add 20 μL RNase-free water to dissolve the RNA, and measure the concentration. cDNA was obtained by reverse transcription according to the instructions of M-MLV Reverse Transcripta...

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Abstract

The invention belongs to the technical field of virus gene engineering, particularly relates to a porcine reproductive and respiratory syndrome mutant virus RvJXwn-nsp1alpha-G90A, and further discloses application thereof. According to the porcine reproductive and respiratory syndrome mutant virus RvJXwn-nsp1alpha-G90A, the 90th amino acid of the non-structural protein nsp1alpha of a high-pathogenicity PRRSV strain JXwn06 is mutated from glycine to alanine to obtain the mutant virus, and a mutant strain capable of inducing and degrading the SLA-I molecular function damage on the surface of the target cell-porcine alveolar macrophages (PAMs) is obtained. After a host cell is infected with the mutant virus, the function of degrading SLA-I molecules on the surface of a target cell can be induced to be seriously damaged, and the mutant virus is stable in hereditary property expression, can be used as a vaccine candidate strain of the porcine reproductive and respiratory syndrome, is used for preventing the porcine reproductive and respiratory syndrome, and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of virus genetic engineering, in particular to a porcine reproductive and respiratory syndrome mutant virus RvJXwn-nsp1α-G90A, and further discloses its construction method and application. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Stage pigs are characterized by respiratory disease. In 1987, the disease first occurred in the United States, and then quickly spread to major pig farming regions around the world. The PRRS epidemic occurred in my country in the mid-1990s and the pathogen was identified, and then PRRSV quickly spread to most pig farming areas in my country, becoming one of the important pathogens affecting the healthy development of pig farming in my country. In 2006, Highly pathogenic PRRSV (Highly pathogenic PRRSV, HP-PRRSV) appeared in my country, whi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/40C12N15/85A61K39/12A61P31/14C12R1/93
CPCC12N7/00C12N15/85C07K14/005A61K39/12A61P31/14C12N2770/10021C12N2770/10022C12N2770/10034
Inventor 杨汉春刘园园韩军郭鑫周磊盖新娜张永宁
Owner CHINA AGRI UNIV
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