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Method for separating and culturing virus of porcine pseudorabies

A technique for porcine pseudorabies and virus inoculation, which is applied in the field of separation and cultivation of porcine pseudorabies virus, and can solve the problems of difficult separation and cultivation of porcine pseudorabies virus, low virus titer, cell death and shedding, etc.

Active Publication Date: 2019-09-27
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cell lines such as rabbit kidney cells and pig kidney cells have been used in my country for decades, and the morphology of the cells has changed significantly due to excessive use. Sexual pathogens are often introduced into these cell lines, making these passaged cells difficult to isolate and culture for porcine pseudorabies virus
Moreover, once these cells are infiltrated with exogenous virus, the cells will die and fall off when the viral titer of PRV does not reach the predetermined titer during the culture process, resulting in the low viral titer of PRV all the time.

Method used

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  • Method for separating and culturing virus of porcine pseudorabies
  • Method for separating and culturing virus of porcine pseudorabies
  • Method for separating and culturing virus of porcine pseudorabies

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Isolation and cultivation of porcine pseudorabies virus SD2011-03 strain

[0029] 1) Disease material collection: select the tonsil, spleen, lymph node or brainstem tissue of pigs with porcine pseudorabies virus positive proved by PCR method. 2) Disease material treatment: Grind about 1 g of disease material with 5 mL of sterile DMEM solution containing antibiotics at a grinding temperature of 0-4°C; then freeze and thaw the grinding liquid at -70°C for 3 times, and centrifuge at 10,000 rpm for 5 Minutes, take the supernatant, discard the precipitate, take the supernatant to extract the total RNA, and perform RT-PCR amplification. A positive sample can be amplified to obtain a specific band with a size of 346bp, such as figure 1 shown. The supernatant of the disease material tested as positive was filtered and sterilized with a 0.22 μm sterile filter, and the supernatant was prepared and stored below -70°C for later use.

[0030] 3) Inoculate 1 mL of the sup...

Embodiment 2

[0039] The PCR identification of embodiment 2 porcine pseudorabies virus

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Abstract

The invention relates to a method for separating and culturing a virus of porcine pseudorabies with a PIEC (Pig Iliac Endothelium Cell), and belongs to the technical field of veterinary biology. According to the method, the virus of porcine pseudorabies is separated and cultured by utilizing the PIEC, and the virus titer of a PRV (Pesudorabies Virus) can reach 108.5-109.0 TCID50 / mL after multiple rounds of purification and multiplication with plaques. The method has the advantages of laying the foundation and providing the technical support for the development of a vaccine, the research on biological specificity, the pathogenic mechanism and the like of the virus of porcine pseudorabies.

Description

technical field [0001] The invention belongs to the field of veterinary biological technology, and in particular relates to a method for isolating and cultivating porcine pseudorabies virus. Background technique [0002] Pseudorabies virus (PRV) is a herpes virus that causes fever, severe itching (except pigs) and encephalomyelitis as the main symptoms of cattle, sheep, pigs, dogs and cats and other domestic and wild animals. Porcine pseudorabies virus is also known as porcine herpesvirus type Ⅰ, infectious bulbar paralysis virus, scrapie virus, and Oyezki's disease virus. Because the clinical symptoms of this disease are similar to rabies, the disease name of "pseudorabies" is used. Porcine pseudorabies virus can cause abortion and stillbirth in pregnant sows, infertility in boars, mass death of newborn piglets, dyspnea and growth stagnation in fattening pigs. It is one of the major infectious diseases that endanger the global pig industry. PRV is a double-stranded DNA vi...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12Q1/70C12Q1/686C12R1/93
CPCC12N7/00C12N2710/16721C12Q1/686C12Q1/701C12Q2563/107
Inventor 张志李晓成李阳刘爽吴发兴张慧张锋王永玲
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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