Recombinant porcine pseudorabies virus TK/gE double-gene deletion strain

A porcine pseudorabies virus and recombinant virus technology, applied in virus/bacteriophage, antiviral agents, recombinant DNA technology, etc., can solve the problems of high price of imported vaccines and unsatisfactory quality of domestic vaccines

Active Publication Date: 2013-01-23
GUANGDONG WENS DAHUANONG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are several porcine pseudorabies gene-deficient vaccines in the domestic market, the price of imported vaccines is high, while the quality of domestic vaccines is not satisfactory. The market demand for high-quality and inexpensive domestic vaccines is extremely urgent

Method used

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  • Recombinant porcine pseudorabies virus TK/gE double-gene deletion strain
  • Recombinant porcine pseudorabies virus TK/gE double-gene deletion strain
  • Recombinant porcine pseudorabies virus TK/gE double-gene deletion strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Isolation and Identification of Porcine Pseudorabies Virus GDXX Strain

[0043] 1 Materials and methods

[0044] 1.1 Test material

[0045] In June 2007, a suspected case of pseudorabies infection occurred in a pig farm in Xinxing County, Guangdong Province. We extracted the brain tissue of the sick pig from the suspected disease material;

[0046] PRV-positive serum, PRV-negative serum, virulent PRV (GDXX strain), and BHK21 cells are preserved by our laboratory.

[0047] 1.2 Disease material treatment

[0048] Aseptically take the diseased pig brain and PBS (0.1M, pH7.2) to make a homogenate at 1:5 (V / V), freeze and thaw 3 times, centrifuge at 3000rpm for 15min, take the supernatant and add double antibody, 37℃ After 18 hours of action, the disease material filtrate was obtained by sterilizing with a 0.45um filter membrane, and stored for future use.

[0049] 1.3 Bab1 / c mouse inoculation test and results

[0050] Take 10 6-week-old Ba1b / c mice, 4 of them...

example 2

[0073] Example two porcine pseudorabies virus GDXX strain TK / gE

[0074] Double gene deletion strain PRV / TK - / gE - The construction and biological characteristics of

[0075] The applicant used the GDXX strain of porcine pseudorabies virus (PRV-GDXX) as the female parent, and sequentially deleted the TK and gE genes by means of molecular biology, and constructed a strain of recombinant pseudorabies virus PRV / TK - / gE - , and systematically studied some biological characteristics of the recombinant virus. The test results are now reported as follows:

[0076] 1. Materials and methods

[0077] 1.1. Materials

[0078] 1.1.1. Viruses and cells

[0079] Porcine pseudorabies virus GDXX strain (PRV-GDXX), BHK 21 Cells and ST cells are preserved by our laboratory.

[0080] 1.1.2. Plasmids and strains

[0081] pcDNA3 / EGFP was donated by Dr. Cheng Jiasen of Sun Yat-sen University; pUC19 vector and DH5α strain were preserved by our laboratory.

[0082] 1.1.3. Tool enzymes and...

example 3

[0214] Example three recombinant virus PRV / TK - / gE - Minimum Immunization Dose Study

[0215] 1. Materials and methods

[0216] 1.1. Vaccine PRV / TK developed in our laboratory - / gE - Gene deletion vaccine, each vial contains 10 viruses 7.0 TCID 50 .

[0217] 1.2. Experimental animals 1-day-old piglets, 70-day-old fattening pigs and sows were negative for pseudorabies virus antibodies.

[0218] 1.3. Experimental plan

[0219] Dilute the developed gene deletion vaccine with diluent so that the virus content per milliliter is 10 6.0 TCID 50 、10 5.0 TCID 50 and 10 4.0 TCID 50 3 different concentrations, and then intramuscularly inoculate 1-day-old piglets, fattening pigs and sows respectively, 8 heads in each group, 1ml for each head. Four weeks after the first immunization, a booster immunization was given with the same dose. Blood was collected 4 weeks after the second immunization to determine the neutralizing antibody and determine the minimum immunization dos...

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Abstract

The invention separates a porcine pseudorabies virus GDXX strain, and a separating strain is served as a female parent for deleting a thymidylate kinase (TK) gene and a gE gene through a molecular biology means in sequence, thus obtaining recombinant virus PRV / TK- / gE-. The recombinant virus provided by the invention is used as a vaccine strain for preparing safe and efficient porcine pseudorabies live vaccine.

Description

technical field [0001] The present invention relates to the technical field of pseudorabies virus, in particular to a recombinant porcine pseudorabies virus TK / gE double-gene deletion strain and a preparation method thereof, and the use of the recombinant porcine pseudorabies virus TK / gE double-gene deletion strain Genetically engineered vaccines. Background technique [0002] Pseudorabies virus (Psedorabies virus, PRV) belongs to herpes virus (Herpesviridac) α herpes virus subfamily (Alphaher-pesririnae) varicella virus (Varicello virus) type I (Porcine herpesvirus Type I), can cause severe disease in pigs. At the same time, pigs are not only the reservoir of pseudorabies virus, but also the source of infection of pseudorabies virus. The control of porcine pseudorabies is not only of great significance to the pig industry itself, but also to the breeding of other animals. Vaccination is the fundamental measure to prevent, control and eliminate pseudorabies. [0003] Early...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/09C12N15/63A61K39/245A61P31/22C12R1/93
Inventor 邵定勇黄红亮陈瑞爱韩静芳唐兆新谢小雨任常宝梁桂益
Owner GUANGDONG WENS DAHUANONG BIOTECH
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