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Type-B avian infectious coryza subunit vaccine as well as preparation method and application thereof

A chicken infectious rhinitis and subunit vaccine technology, which is applied in the directions of botanical equipment and method, biochemical equipment and method, application, etc., can solve the problems of high culture cost, side reactions, focal necrotic spots, etc. Simple process, low cost, excellent immune effect

Inactive Publication Date: 2020-11-17
乾元浩生物股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because Aviobacterium paragallinarum is a fastidious bacterium, the cost of cultivation is relatively high, and the inactivated vaccine contains a large amount of bacterial protein, endotoxin, other toxic substances and additives, which may easily cause serious side effects. Focal necrotic plaques also form at the inoculation site

Method used

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  • Type-B avian infectious coryza subunit vaccine as well as preparation method and application thereof
  • Type-B avian infectious coryza subunit vaccine as well as preparation method and application thereof
  • Type-B avian infectious coryza subunit vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Optimization of B-HMTp210-R2 nucleotide sequence

[0027] In order to improve the expression level, soluble expression level and stability of target protein, the present embodiment is to the nucleotide sequence (as shown in SEQ ID NO.3) and amino acid of B-type chicken infectious rhinitis outer membrane protein B-HMTp210-R2 The sequence has been optimized in many ways. After a large number of screening and comparative experiments, the present invention finally determines the optimization scheme of the B-HMTp210-R2 nucleotide sequence (the optimized nucleotide sequence is shown in SEQ ID NO.2):

[0028] 2. Soluble expression of B-HMTp210-R2 protein

[0029] The above-mentioned B-HMTp210-R2 gene sequence optimized by nucleotide sequence was synthesized, and B-HMTp210-R2 before and after nucleotide sequence optimization were respectively digested with Nde I / Xho I and cloned into the pET-30a vector, The obtained recombinant expression vectors were named pET30a-B-HMTp21...

Embodiment 2

[0031] The present embodiment provides a preparation process of type B chicken infectious rhinitis subunit vaccine, and the specific steps are as follows:

[0032] 1. Expression of B-HMTp210-R2 protein:

[0033] (1) Activation of genetically engineered bacteria, get 10 μL of engineered bacteria pET30a-B-H MTp210-R2-OPTI glycerolbacteria constructed in Example 2 and inoculate in 5ml of kana-resistant LB medium, culture overnight with shaking;

[0034] (2) Inoculate 200mL of Kanna-resistant culture medium at a ratio of 1:200, culture with shaking at 37°C for 4-5 hours, until the OD600 value reaches about 0.8;

[0035] (3) Add IPTG at a ratio of 1:2000, and induce at 16°C for 15 hours;

[0036] (4) collect thalline;

[0037] (5) Resuspend bacteria with 20mL special buffer;

[0038] (6) Ultrasonic disruption of bacteria;

[0039] (7) 12000rpm centrifugal 10min, centrifugal 2 times;

[0040] (8) Add 40% ammonium sulfate to precipitate the target protein, centrifuge at 12000rpm...

Embodiment 3

[0057] The present embodiment carries out efficacy analysis to the type B chicken infectious rhinitis subunit vaccine prepared in embodiment 2, specifically as follows:

[0058] (1) The safety test of the minimum age of use and a single-dose inoculation by different routes

[0059] Divide 70 7-day-old SPF chickens into 4 groups, with 20 chickens in the immunization group 1-3, 10 chickens in the control group in the 4th group, and 10 chickens in the control group in the 1-3 immunization group. Each group is further divided into two groups, with 10 SPF chickens in each group Chickens were inoculated with Type B chicken infectious rhinitis subunit inactivated vaccines (batches were 20190712, 20190805 and 20190820 respectively, the preparation method was the same as in Example 2) by different immunization routes (intramuscular injection or neck subcutaneous injection), and the dose was 0.5mL / bird; 10 SFP chickens in the control group were subcutaneously injected with sterilized ph...

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Abstract

The invention relates to a type-B avian infectious coryza subunit vaccine as well as a preparation method and application thereof. Region2 (B-HMTp210-R2) of a type-B avian infectious coryza outer membrane protein HMTp210 gene is adopted as an immunogen to prepare the type-B avian infectious coryza subunit vaccine,and a nucleotide sequence of the type-B avian infectious coryza is optimized. The optimized nucleotide sequence is cloned into a PET-30a expression vector, and BL21 host bacteria are transformed, so that the soluble expression quantity of B-HMTp210-R2 is remarkably improved by two times or more, and the stability is remarkably improved. The prepared type-B avian infectious coryza subunit vaccine has higher safety and immune effects, can quickly induce immune animals to generate high-level neutralizing antibodies, and realizes efficient immune protection against type-B avian infectious coryza. The subunit vaccine is simple in preparation process, low in cost and easy for large-scale production.

Description

technical field [0001] The invention relates to the field of veterinary biological products, in particular to a type B chicken infectious rhinitis subunit vaccine and its preparation method and application. Background technique [0002] Avian infectious coryza is an acute upper respiratory infectious disease of chickens caused by Avibacterium paragallinarum (Apg) infection. At present, the disease has caused various problems in the field of chicken farming, especially causing a great impact on the production performance of laying hens. The disease mainly causes poor growth of rearing chickens and a significant drop in egg production of laying hens, and can significantly increase the elimination rate of broiler chickens, causing great economic losses. [0003] Paragallinia is a short Gram-negative bacillus of the Pasteurella family. Page et al. divided A. paragallinia into three serotypes, A, B, and C. Later studies showed that there were three serotypes of A, B, and C. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/102A61K39/39A61P31/04C12N15/31C07K14/285
CPCA61K39/102A61K39/39A61P31/04C07K14/285A61K2039/55511A61K2039/523A61K2039/552
Inventor 王增福李跃魏杰吴宗学赵成全程海波李海鹰倪娇史张艳
Owner 乾元浩生物股份有限公司
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