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Pig pseudorabies virus natural low-virulent C strain and heatproof preservation method thereof

A technology of porcine pseudorabies virus and preservation method, which is applied in the field of natural attenuated strain C of porcine pseudorabies virus and its heat-resistant preservation, can solve the problems of restricting the overseas market expansion of domestic vaccines, impacting the domestic vaccine market, and affecting the development of biological products enterprises, etc. , to achieve good stability

Active Publication Date: 2012-04-04
SHANGHAI CHUANG HONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the storage temperature of live pseudorabies vaccines in my country is still -15°C, and imported vaccines have caused a huge market impact on domestic vaccines, affecting the development of domestic biological products companies.
The backwardness of technology also limits the expansion of overseas markets for domestic vaccines

Method used

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  • Pig pseudorabies virus natural low-virulent C strain and heatproof preservation method thereof
  • Pig pseudorabies virus natural low-virulent C strain and heatproof preservation method thereof
  • Pig pseudorabies virus natural low-virulent C strain and heatproof preservation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Isolation and identification of strain C: collect brain, heart, liver, spleen, lung, kidney and other disease materials from aseptic samples, and store them in a -20°C refrigerator for later use. Aseptically take the diseased pig brain and PBS (0.1M, pH7.2) to make a homogenate at 1:5 (V / V), freeze and thaw three times at 3000r / min, centrifuge for 15min, take the supernatant and add double Anti-bacteria, 37 ° C for 18 hours, sterilized by 0.45 μm filter membrane. Inoculation 0.5 mL The virus filtrate was grown into a monolayer of PK-15 cells and observed continuously until cytopathic changes (CPE) appeared. The strain was obtained by cloning and purification.

[0032] Perform 10-fold serial dilutions of PRV negative serum and positive serum respectively (serum concentration is 10 -1 ~10 -10 ). Add the same amount of 200TCID 50 , 37 ℃ sense for 20min. Inoculate 96-well plate monolayer PK-15 cells respectively, 0.1ml per well, inoculate 8 wells for each dilutio...

Embodiment 2

[0043] Heat-resistant Preservation Technology of Pseudorabies Virus

[0044] (1) Formula and preparation method of lyoprotectant

[0045] Heat-resistant ingredients: 1% gelatin, 8% sucrose, 1% EDTA, and then use double distilled water to dissolve to a suitable scale. Autoclave at 121°C for 30 minutes.

[0046] Heat-labile ingredients: 1% monosodium glutamate, 2% dipotassium hydrogen phosphate, 0.52% potassium dihydrogen phosphate, 1% bovine serum albumin, 2% tryptone. Sterilize with a 0.22μm membrane filter.

[0047] Then the two are mixed 1:1.

[0048] (2) After the pseudorabies virus is cultivated, carry out sterility test and determination of virus content, mix the qualified virus liquid and the prepared freeze-drying protective agent 1:1, shake well, and freeze immediately after quantitative subpackaging dry.

[0049] (3) Put the sub-packaged vaccine into the freeze dryer box, slowly cool down to -45°C within 3 hours, maintain at this temperature for 6 hours, start va...

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Abstract

The invention relates to a pig pseudorabies virus natural low-virulent C strain and a heatproof preservation method thereof. The microbe collection registry number of the strain is CCTCC NO: V201114. According to the invention, various cryoprotectant substrates are mixed and blended according to a certain ratio; the cryoprotectants are respectively subject to aseptic processing; cryoprotectant components that can be subject to high-pressure sterilization are dissolved in bi-distilled water, and are sterilized for 15-30min under a temperature of 108-121 DEG C; cryoprotectant components that can not be subject to high-pressure sterilization are dissolved in bi-distilled water according to a certain formula, and are sterilized by using a filter membrane with a size of 0.22mum; all the cryoprotectant components are then mixed into a heatproof cryoprotectant; the heatproof cryoprotectant is mixed with a pseudorabies virus liquid according to a ratio of 1:1-1.2; and the mixture is lyophilized with a corresponding lyophilization curve. Compared to prior arts, the C strain provided by the invention has good stability. In a fifth generation animal of continuous back-procreation, the genetic sequence is stable, and no mutation is found. Therefore, the strain provided by the invention provides a good resource for the researches of pig pseudorabies low-virulent vaccines. The strain can bepreserved for 24 months under a temperature of 2-8 DEG C, and the virus titer is reduced by no more than 1 titer.

Description

technical field [0001] The invention belongs to the field of live virus vaccine production for animals, and in particular relates to a natural attenuated strain C of porcine pseudorabies virus and a heat-resistant preservation method thereof. Background technique [0002] Pseudorabies virus (Pseudorabies Virus, PRV) is a herpes virus that can cause pseudorabies in a variety of domestic and wild animals. Animals other than pigs showed fatal infection, and pigs were the natural host and storage host of PRV, showing epidemic infection. Adult pigs often present with occult infection and respiratory symptoms, sows with abortion, stillbirth and mummified fetuses, and boars with orchitis. Piglets exhibited fever, coma and neurological symptoms, and mortality was high. Therefore, pseudorabies is an important infectious disease in pig industry. [0003] At present, the attenuated live vaccine is one of the main vaccines for preventing infectious diseases of livestock and poultry. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 赖志漆世华高俊锋
Owner SHANGHAI CHUANG HONG BIOTECH
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