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IBV-III antigen production process

A technology of IBV-HI and production process, applied in the biological field, can solve the problems of long dialysis concentration time, inability to carry out batch production and use, and long time, so as to optimize the preparation process and operation steps, improve reliability and reliability Good operability and stability

Inactive Publication Date: 2011-11-16
秦卓明 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The IBV treated with trypsin has hemagglutination activity, but it is non-specific and is not suitable as a detection antigen; the potency of the IBV-HI antigen prepared by PLC I is the same as that using the culture medium of Clostridium welchii type A in rabbits. The prepared IBV-HI antigen is equivalent, but due to the lack of synergistic effects of other factors in the culture solution of Clostridium welchii type A in rabbits, the production cost is relatively high, which affects the popularization and application of the antigen
Therefore, it is not practical to produce and use it as an antigen for detection, and generally it can only be used as a laboratory test.
However, the cost of culture solution of Clostridium welchii type A in rabbits is low, but when the current technology is used for production, the entire production process takes a long time, and the final antigen has a short validity period, which cannot achieve the purpose of long-term detection and cannot Carry out mass production and practical application
At the same time, in the prior art, the method of dialysis is generally used to concentrate the virus liquid. With this method, the dialysis concentration takes a long time and only a small amount of virus liquid can be concentrated, and it cannot be concentrated in large quantities and in a long time The operation of time will have a certain impact on the virus itself, inactivating part of the virus and reducing the titer of the prepared antigen. Therefore, this method can only be used as a laboratory test and cannot be used in batch production.

Method used

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  • IBV-III antigen production process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] After diluting infectious bronchitis virus (IBV) by 1:1000 times, inoculate 11-day-old SPF chicken embryos, 0.2ml per embryo, continue to incubate at 37°C, light eggs once every 24 hours, discard dead chicken embryos, and then Eggs were illuminated once every 4 hours, and the dead chicken embryos were taken out in time and cooled at 2-8°C. After 96 hours, all eggs were taken out and placed at 2-8°C for 12-16 hours. 100ml of chicken embryo allantoic fluid of type IBV was centrifuged at high speed for 40min at a speed of 5000-6000rpm, and 98ml of supernatant was obtained after centrifugation. The supernatant was then subjected to ultracentrifugation for 2.0 h at a rotational speed of 25000 g, the supernatant was discarded, and the precipitate was suspended with 0.5 ml of 0.01 M Tris-HCl physiological saline. After suspension, culture for 6 hours after inoculation, centrifuge at 2000r / min for 40 minutes, filter and sterilize the obtained rabbit Clostridium welchii type A f...

Embodiment 2

[0017]150 ml of chicken embryo allantoic fluid containing respiratory IBV harvested by common methods was subjected to high-speed centrifugation for 50 minutes at a speed of 9000 to 10000 rpm, and 146 ml of supernatant was obtained after centrifugation. The supernatant was then subjected to ultracentrifugation for 3.0 h at a centrifugation speed of 30000 g, the supernatant was discarded, and the precipitate was suspended in 1.5 ml of 0.1 M PBS buffer of pH 5.9. After suspending, incubate for 12 hours after inoculation, centrifuge at 3000r / min for 35 minutes, filter and sterilize the rabbit Clostridium welchii type A filtrate, mix with equal volumes, and put it in a 4℃ refrigerator for 2.5 hours in a 37℃ water bath After 36 hours, the antigen was obtained by freeze-drying. This product is milky white, loose and porous sponge-like dry goods.

Embodiment 3

[0019] After diluting infectious bronchitis virus (IBV) by 1:1000 times, inoculate 11-day-old SPF chicken embryos, 0.2ml per embryo, continue to incubate at 37°C, light eggs once every 24 hours, discard dead chicken embryos, and then Eggs were illuminated once every 4 hours, and the dead chicken embryos were taken out in time and cooled at 2-8°C. After 96 hours, all eggs were taken out and placed at 2-8°C for 12-16 hours. 200ml of chicken embryo allantoic fluid of type IBV was centrifuged at high speed for 60min at a speed of 7000-8500rpm, and 190ml of supernatant was taken after centrifugation. The supernatant was then subjected to ultracentrifugation for 2.5 h at a rotational speed of 28000 g, the supernatant was discarded, and the precipitate was suspended with 0.2 ml of 0.01 M Tris-HCl physiological saline at pH 6.5. After suspension, culture for 36 hours after inoculation, centrifuge at 4000r / min for 20 minutes, filter and sterilize the obtained rabbit Clostridium welchii...

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Abstract

The invention supplies a manufacturing technique of IBV-HI antigen, the preparation process of antigen is that: the virus fluid of containing the protein virus is isolated from chicken embryo allantois fluid of containing respiratory type IBV the virus and blending with clostridium filtrate of rabbits A type wei surname as a culture medium, after two hours water bath action with 37deg.C, put the fluid into the 4deg.C fridge for 48 hours, through freeze drying to get antigen, the related centrifuge divided into two centrifugal, first the mixed fluid through high speed centrifugation, choose the supernatant after the centrifugation; then it progress the two centrifugation as ultracentrifugation, the time is 2-3h, the rotation rate of centrifugation is 25000-35000g, abandon the supernatant after the centrifugation, and use deposit suspension to obtain virus fluid. Using this invention method, infectious bronchitis HI antigen of respiratory system has relatively low cost, type specificity, high sensitivity, stability feature, it makes HI method to detect respiratory infectious bronchitis feasible, and enhance the credibility and maneuverability of its detection.

Description

technical field [0001] The invention relates to biotechnology, in particular to a production process of IBV-HI (respiratory infectious bronchitis) antigen. Background technique [0002] Chicken infectious bronchitis (Infectious Bronchitis, referred to as IB) is caused by infectious bronchitis virus (Infectious Bronchitis Virus, referred to as IBV) as an acute, highly contagious respiratory and genitourinary tract disease in chickens, characterized by tracheal rales Respiratory symptoms such as coughing, sneezing, kidney swelling, and uric acid deposition in the ureter are the main pathological features, and can also cause swelling of the glandular stomach, enteritis, oviduct cyst, and atrophy of chickens, which are also important causes of "false hens". reason. There are more than 20 serotypes of IBV, and there is no or only partial cross-protection between different serotypes. Respiratory infectious bronchitis is currently the main virus strain in chicken flocks in my cou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/02
Inventor 秦卓明徐怀英欧阳文军王友令贾强袁小远杨金兴张世栋仉伟
Owner 秦卓明
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