Pseudorabies virus and application of pseudorabies virus

A porcine pseudorabies and virus technology, applied in antiviral agents, virus/bacteriophage, inactivation/attenuation, etc., can solve the problems that vaccines cannot completely protect PRV and economic losses of immunized pig farms, and achieve high protection rate and safety Good results

Active Publication Date: 2015-07-29
SHANDONG SINDER TECH
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  • Summary
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  • Application Information

AI Technical Summary

Problems solved by technology

The most widely used vaccine in the world is mainly the PRV Bartha-K61 vaccine. However, in recent years, the epidemic situation of porcine pseudorabies in my country is that the existing vaccine cannot completely protect against the attack of the new epidemic PRV, which has caused certain economic losses to the immunized pig farms. loss

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0009] Embodiment 1: Isolation of porcine pseudorabies virus

[0010] In recent years, pseudorabies has occurred in many pig farms in my country, most of which are breeding pig farms, and the affected pigs have been injected with pseudorabies vaccine before, and it is speculated that the infected virus has mutated; Screening for porcine pseudorabies virus.

[0011] The visceral samples of sick pigs were taken, including: heart, liver, lung, spleen, tonsil, and lymph nodes. Homogenate the visceral sample and PBS (0.1M, pH7.2) at V / V1:5, freeze-thaw 3 times, centrifuge at 3000r / min for 15min, add double antibody to the supernatant, incubate at 37°C for 1h, Sterilize by filtration through a 0.22 μm membrane filter. Take 1ml of the virus filtrate and inoculate the Vero cells grown into a single layer, pass three generations blindly, and observe the cytopathic changes (CPE). The cell culture medium with CPE was plaque-purified, and the purified virus was subpackaged and stored at...

Embodiment 2

[0012] Embodiment 2: the breeding and identification of the porcine pseudorabies virus of screening

[0013] 1. Cultivation The porcine pseudorabies virus that was screened was named PRV QD strain, which was identified as virulent by PCR, domesticated by Vero cell culture, and the virus content reached 10 7.0 TCID 50 / More than 0.1ml. The virus was deposited on March 6, 2015 at the General Microbiology Center of the China Committee for the Collection of Microbial Cultures at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.10266.

[0014] 2. Identification

[0015] 2.1 Primer design Referring to the published PRV gE gene fragments, a pair of primers were designed using Oligo 7.0 to amplify the full length:

[0016] F1: 5'-ATGCGGCCCTTTCTGCTGCGCGCC-3';

[0017] F2: 5'-TTAAGCGGGGCGGGACATCAACAGGC-3'.

[0018] 2.2 Identification results PCR detection was performed on...

Embodiment 3

[0024] Embodiment 3: Preparation of antigen for seedling production

[0025] The DNA of the isolated strain was extracted from the clinically isolated porcine pseudorabies virus, and the virulence genes TK, gE and gI genes of the isolated strain were sequentially deleted by genetic engineering methods, and named PRV / TK - / gE - / gI - Poisonous seeds, multiplied and poisonous, can be used as seed poison after adding protective agent.

[0026] PRV / TK - / gE - / gI - Virus seed safety test: Dilute the antigen 10 times with PBS, inoculate four 100g mice intramuscularly, 0.2mL each, observe for 14 days, no more than two of them react or die. The results showed that the vaccine was safe for mice, had no pseudorabies-specific symptoms, and did not affect growth and development.

[0027] The above-mentioned virus seed safety test result illustrates that the porcine pseudorabies virus gene deletion vaccine (PRV / TK) of the present invention's transformation - / gE - / gI - ) is sa...

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Abstract

The invention provides a pig pseudorabies virus and the application of the pig pseudorabies virus. The preservation number of the pig pseudorabies virus is CGMCC No.10266. The selected pig pseudorabies virus has the characteristics of high safety and high protection rate. A pig pseudorabies virus PRV-QD strain is inoculated with a Vero cell to form cell venom, the DNA (deoxyribonucleic acid) of the strain is extracted, the separated strain is subjected to deletions of virulence gene TK, gE and gI gene by a genetic engineering method, the virus is propagated after cell retrieval, and after a protecting agent is added into the virus, a vaccine is formed. The protection rate of the manufactured bacterin to piglets is 100 percent, the efficiency is obviously more excellent than that of a Bartha-K61 group (20 percent), and the vaccine has the advantages of high efficiency and high safety.

Description

technical field [0001] The invention belongs to the technical field of livestock disease source microorganism screening, and in particular relates to a pseudorabies virus and its application in preparing vaccines. Background technique [0002] Porcine pseudorabies virus (Pseudorabies virus, PRV) belongs to herpesviridae a herpesvirinae porcine herpesvirus type I, pigs are the only natural host of the virus, causing pig pseudorabies (Pseudorabies, PR). The disease is mostly outbreaks in pig herds, mainly harming sows, causing abortion or vertical transmission to piglets, resulting in a large number of deaths of newborn piglets, which has brought huge economic losses to the pig industry in my country and even the world. At present, there is no effective drug for the treatment of PR, so vaccination has become the main measure to control the occurrence and prevalence of the disease. The most widely used vaccine in the world is mainly the PRV Bartha-K61 vaccine. However, in rece...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/04A61K39/245A61P31/22C12R1/93
Inventor 李明义刘杉杉孙伟刘阳李晓林赵航李彦凤葛栋李佳琪
Owner SHANDONG SINDER TECH
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