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Nadc30-like pig PRRS strong virus strain, weak virus strain and application thereof

A porcine PRRS virus, a technology for porcine PRRS, which is applied in the application field of preventing NADC30-like porcine reproductive and respiratory syndrome, can solve problems such as the protective effect of pigs, and achieve the effect of good protection effect and high safety.

Active Publication Date: 2022-05-10
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that although there are many kinds of PRRS vaccines, none of them can produce enough protection for pigs.

Method used

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  • Nadc30-like pig PRRS strong virus strain, weak virus strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Isolation and identification of embodiment 1 class NADC30 pig PRRS virulent strain SD strain

[0026] Organs were collected from sick pigs in a farm with "massive abortion and piglet emaciation" in Shandong Province, my country. Grind the collected disease material, centrifuge, filter, and freeze the filtrate for later use. After culturing Marc-145 cells at 37°C for 48-72 hours to form a monolayer, inoculate 1ml of the filtered disease material suspension supernatant in each bottle, and absorb 2 ~4 hours later, change to DMEM culture medium containing 2% fetal bovine serum and continue culturing at 37°C, and observe whether there is cytopathic disease (CPE) in 4 to 5 days. When 60-70% of the cells have CPE changes, the virus is collected, and the virus strain with the disease is passed on for 2 to 3 generations, and then PCR amplification is performed with Nsp2 and ORF5 primers of PRRSV; N and M-specific monoclonal antibodies of the PRRSV HuN4-F112 strain are used Iden...

Embodiment 2

[0028] The cultivation of embodiment 2 class NADC30 pig PRRS attenuated strain SD-R strain

[0029] 1. Material method

[0030] 1.1 Virus cell culture and cloning

[0031] Marc-145 cells for virus cultivation were cultured with DMEM cell culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Use this cell culture medium to resuscitate Marc-145 cells or pass them to a sterile cell culture flask with a capacity of 100ml, and use them for virus inoculation after they grow into a good cell monolayer.

[0032] 1.2 Cloning of viruses

[0033] The NADC30 porcine PRRSV SD strain isolated in Example 1 of the present invention is passed on Marc-145 cells, and virus plaques are cloned when passing on to the 10th, 15th, and 20th generations respectively, and plaques with a diameter of 1 mm are selected to continue Subculture.

[0034] 1.3 Determination of Poison Value of Toxins

[0035] Select different generations of seed virus to determine TCID according to ...

Embodiment 3

[0043] Preparation of embodiment 3 class NADC30 porcine reproductive and respiratory syndrome virus attenuated vaccine

[0044] Select the well-grown Marc-145 cells for use, after pouring out the culture solution, replace it with a fresh culture solution (DMEM culture solution) containing 1% v / v virus seed solution (the attenuated strain PRRSV SD-R that embodiment 2 obtains), place Culture was continued at 37°C. Observe daily. Cells with abnormal growth and contamination should be discarded. After 2 to 3 days of culture, about 80% of the cells have lesions, and when the difference between the control cells is significant, they can be harvested and stored at -20°C after 3 times of freezing and thawing. Sampling the harvested virus for sterility test should show no bacterial growth. Potency should be ≥ 10 6.5 TCID 50 / ml. Mix the qualified virus liquid with sucrose gelatin protective agent (280g of sucrose, 49g of gelatin and 700ml of deionized water after heating and diss...

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Abstract

The invention discloses strong and weak strains of NADC30-like porcine PRRS and applications thereof. The virulent NADC30-like porcine PRRS virus strain of the present invention is named as NADC30-like porcine PRRS virus SD strain, and its microorganism preservation number is: CGMCC NO.19691. By attenuating the SD strain, the present invention has also obtained a NADC30-like attenuated strain of porcine PRRS virus, named after the NADC30-like porcine PRRS virus SD-R strain, and its microorganism preservation number is: CGMCC NO.19692 . Experiments have proved that the SD strain of the present invention has the characteristics of strong virulence and stable pathogenicity to piglets, and can be used for testing, preventing or treating porcine reproductive and respiratory syndrome of class NADC30; the attenuated vaccine strain SD-R strain of the present invention has safety High and good protective effect, it can be used to prevent NADC30-like porcine reproductive and respiratory syndrome. Therefore, the SD strain of the present invention and its attenuated vaccine SD-R strain can be used to prepare a single vaccine or combined vaccine (live or inactivated vaccine) for preventing and treating NADC30 porcine reproductive and respiratory syndrome, and can also be prepared as a diagnostic NADC30 vaccine. Diagnostic reagents for porcine reproductive and respiratory syndrome.

Description

technical field [0001] The invention relates to a strong and attenuated strain of NADC30-like porcine PRRS, and also relates to the application of the attenuated strain in preventing NADC30-like porcine reproductive and respiratory syndrome. The invention belongs to the field of biomedicine. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as porcine reproductive and respiratory syndrome (PRRS), is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), characterized by sow abortion and piglet respiratory symptoms. . In 1996, PRRSV was reported for the first time in my country, and was later classified as a classic strain of my country. Subsequently, the classic porcine reproductive and respiratory syndrome vaccine virus was widely used in my country, and the prevalence of PRRSV in my country was well controlled. In 2006, "high fever" of pigs broke out in the southern part of my country...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N7/08A61K39/12A61K47/42A61K47/26A61K9/19A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61K47/42A61K47/26A61K9/19A61P31/14C12N2770/10021C12N2770/10064C12N2770/10034A61K2039/5254A61K2039/552
Inventor 田志军张洪亮蔡雪辉相丽润王倩汤艳东彭金美安同庆
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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