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
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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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