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Preparation method of chicken infectious bronchitis virus HA antigen

A bronchitis and chicken infectious technology, applied in the field of bioengineering, can solve the problems of low ultracentrifugation antigen titer, high price of phospholipase C, and increased cost of HA antigen solution, so as to solve inaccurate and stable judgment results , Judgment results are more effective

Inactive Publication Date: 2012-03-28
哈药集团生物疫苗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the production methods of HA antigen solution are still concentrated by ultracentrifugation, treated with phospholipase C, and inactivated by commonly used inactivating agents, adding chitosan as a stabilizer, etc. The main problems of the original method are: ( 1) Due to the high price of phospholipase C, the cost of HA antigen solution is increased
(4) Ultracentrifugation antigen titer is low

Method used

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  • Preparation method of chicken infectious bronchitis virus HA antigen
  • Preparation method of chicken infectious bronchitis virus HA antigen
  • Preparation method of chicken infectious bronchitis virus HA antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The preparation of embodiment 1N-2-hydroxypropyltrimethylammonium chloride chitosan

[0037] method:

[0038] (1) Deacetylation treatment of chitosan

[0039] Disperse 50 g of chitosan in a solution with a mass fraction of 20% NaOH, reflux and stir for 3 hours at 110° C. and a pressure of 0.2 Mpa, pour off the supernatant, and wash with deionized water until neutral;

[0040] (2) soaking treatment of chitosan

[0041] Dissolve 2 g of deacetylated chitosan in 1% acetic acid solution by volume fraction, stir to dissolve, and vacuum filter to remove insoluble matter. Add 0.1mol / L NaOH solution drop by drop under the stirring condition of 800r / min, adjust the chitosan solution to pH ≈ 9, gradually a white precipitate precipitates, soak for 8 hours, filter with suction, wash with deionized water until the filtrate is neutral, and drain the water; Finally, add 15 mL of isopropanol to the filter cake, stir for 30 minutes to disperse evenly, and pour it into a three-necked f...

Embodiment 2

[0046] The preparation of embodiment 2 chicken infectious bronchitis virus HA antigen

[0047] method:

[0048] (1) Virus proliferation

[0049] After diluting chicken kidney type infectious bronchitis virus M41 strain with sterile normal saline at a volume ratio of 1:50, inoculate 11-day-old SPF chicken embryos through the allantoic cavity, 0.1 mL per embryo, continue incubation at 37°C, discard Remove the dead embryos before 24 hours. After 36 hours, take out all the embryos and put them in the refrigerator at 4°C overnight. Collect the allantoic fluid and store them at 4°C for later use.

[0050] (2) Concentration of virus liquid and treatment of culture liquid of Clostridium perfringens type A

[0051] Clostridium perfringens culture solution was prepared according to the following method: the Clostridium perfringens strain was inoculated in the anaerobic broth medium with an inoculation amount of 3%, and then placed in a 37° C. incubator for 24 hours. The culture solut...

Embodiment 3

[0058] Example 3 Antigen Potency Test

[0059] Use the chicken infectious bronchitis live vaccine product produced by our company to collect serum after immunizing chickens according to the routine immunization procedure, and use the three batches of antigens made by ourselves according to the method in Example 2 for efficacy testing. Five chicken sera were collected, treated with kaolin according to conventional methods, and tested for antibody detection by hemagglutination inhibition test. The results are shown in the following table 1-table 3:

[0060] Table 1 Antigen lot number: 2011001

[0061]

[0062] Table 2 Antigen lot number: 2011002

[0063]

[0064] Table 3 Antigen lot number: 2011003

[0065]

[0066] From the above results, it can be known that the IBV HA antigen prepared by the present invention can be successfully applied to the detection of serum HI antibody titer after chicken IBV vaccine immunization.

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Abstract

The invention discloses a preparation method of a chicken infectious bronchitis virus HA antigen. In the preparation method, an A-type clostridium perfringen is adopted to treat a virus solution, so that the problem that in the conventional method, the preparation cost of the antigen is high is solved; an adopted stabilizing agent is N-2-hydroxypropyl trimethyl ammonia chloride chitosan and is water-soluble, so that the problem that the conventional stabilizing agent chitosan is water-insoluble is solved; an adopted deactivating method is deactivation for 30 minutes in water bath of 56 DEG C, so that the problem that the titer is reduced after a deactivating agent is added is solved; an adopted chicken red blood cell suspension has the concentration of 0.5 percent, so that the judgment result is more stable and the problem that the judgment result is not accurate is solved; due to adoption of dialysis bag for concentration, the problem that the titer of the antigen is low is solved; therefore, the invention provides the preparation method of the chicken infectious bronchitis virus HA antigen, which is low in cost, high in titer, and high in stability and safety, and the prepared IBV (infectious bronchitis virus) HA antigen can be successfully applied to detection on the titer of a serum HI antibody after chickens are immunized by an IBV vaccine.

Description

technical field [0001] The invention relates to a preparation method of an antigen, in particular to a preparation method of chicken infectious bronchitis virus HA antigen. in the field of bioengineering, Background technique [0002] Chicken infectious bronchitis (IB) is an acute highly contagious disease caused by chicken infectious bronchitis virus (IBV). Symptoms include coughing, sneezing, and tracheal rales. The egg production of laying hens decreased, and deformed eggs were produced, and the performance of young chickens was particularly serious. Certain strains cause kidney and intestinal disease. Chicken infectious bronchitis has caused serious economic losses to the world poultry industry. Due to the high variability of IBV, there are many serotypes, and the cross-protective reaction between various serotypes is low, and the vaccine immunization often loses the expected effect. [0003] Most IBVs have no agglutinogen on the surface and have no natural hemagglu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165
Inventor 孙德君丁国杰张扬王晶赵辉
Owner 哈药集团生物疫苗有限公司
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