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Group I serum type 4-serum type 8 avian adenovirus bivalent subunit vaccine and preparation method thereof

A subunit vaccine and poultry adenovirus technology, applied in botany equipment and methods, biochemical equipment and methods, antiviral agents, etc., to achieve the effects of easy large-scale production, efficient immune protection, and simple preparation process

Active Publication Date: 2019-05-07
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

At present, there is no report of serotype 4-serotype 8 avian adenovirus bivalent subunit inactivated vaccine

Method used

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  • Group I serum type 4-serum type 8 avian adenovirus bivalent subunit vaccine and preparation method thereof
  • Group I serum type 4-serum type 8 avian adenovirus bivalent subunit vaccine and preparation method thereof
  • Group I serum type 4-serum type 8 avian adenovirus bivalent subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The fiber protein sequence analysis of embodiment 1 poultry adenovirus strain QYHFADV-4 and QYHFADV-8

[0059] The avian adenovirus strains QYHFADV-4 and QYHFADV-8 isolated from chickens suffering from adenoviral disease were amplified by PCR to obtain the fibrin coding gene, and the nucleotide sequence of the coding gene was obtained by sequencing and analysis. The amino acid sequence of its corresponding protein, wherein, the amino acid sequence of the fibrin of avian adenovirus strain QYHFADV-4 is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.3; The amino acid sequence of the fibrin of virus strain QYHFADV-8 is shown in SEQ ID NO.2; the nucleotide sequence of the coding gene is shown in SEQ ID NO.5. Sequence homology and phylogenetic tree analysis were performed on the fibrin derived from QYHFADV-4 and QYHFADV-8 obtained above and the avian adenovirus fibrin disclosed in the prior art. The sequence homology of known avian...

Embodiment 2

[0060] Example 2 Soluble expression of serotype 4 and serotype 8 avian adenovirus fibrin

[0061]In order to improve the expression amount and soluble expression level of fibrin, at first the fibrin coding gene (as shown in SEQ ID NO.3) of serum type 4 avian adenovirus strain QYHFADV-4 and serum type 8 avian adenovirus strain QYHFADV The fibrin-encoding gene of -8 (as shown in SEQ ID NO.5) was codon-optimized. Referring to the codon preference of E. coli host bacteria, after a large number of screening and comparison experiments, the codon optimization of the fibrin gene of QYHFADV-4 was finally determined as follows (the optimized synonymous codons are in brackets): 6 arginines Sites, respectively 4 AGA (CGC), 1 AGG (CGC) and CGA (CGC) (respectively corresponding to the 7th, 8th, 34th, 85th, 338th and 440th positions of SEQ ID NO.1); Leucine 6 sites, CTA (CTG) (respectively corresponding to the 65th, 210, 231, 255, 264 and 444th positions of SEQ ID NO.1); 16 proline sites CC...

Embodiment 3

[0066] Example 3 Preparation of serotype 4-serotype 8 avian adenovirus bivalent subunit vaccine

[0067] 1. Expression of fibrin antigen of serotype 4 and serotype 8 avian adenovirus QYHFADV-4 and QYHFADV-8:

[0068] (1) Activation of genetically engineered bacteria, take 10 μL of glycerol bacteria of the engineering bacteria PET30a-QYHFADV-4 fiber and PET30a-QYHFADV-8 fiber constructed in Example 2 and inoculate them in 5ml of kana-resistant LB medium, and culture overnight with shaking;

[0069] (2) Inoculate 200mL of Kanna-resistant medium at a ratio of 1:200, culture with shaking at 37°C for 4-5h, until OD 600 The value reaches about 0.8;

[0070] (3) Add IPTG at a ratio of 1:2000, and induce at 16°C for 15 hours;

[0071] (4) collect thalline;

[0072] (5) Resuspend bacteria with 20mL special buffer;

[0073] (6) Ultrasonic disruption of bacteria;

[0074] (7) 12000rpm centrifugal 10min, centrifugal 2 times;

[0075] (8) SDS-PAGE electrophoresis detection, analysis ...

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Abstract

The invention discloses a group I serum type 4-serum type 8 avian adenovirus bivalent subunit vaccine and a preparation method thereof. The vaccine comprises type-4 avian adenovirus fibrin with an amino acid sequence as shown in SEQ ID NO.1 and type-8 avian adenovirus fibrin with an amino acid sequence as shown in SEQ ID NO.2. The method adopts the full-length fibrin of avian adenovirus serum type4 and serum type 8 as an immunogen to prepare the bivalent subunit vaccine, and after optimization by a specific codon optimization mode, the expression quantity and the soluble expression quantity of serum type 4-serum type 8 fibrinare obviously improved. The bivalent subunit vaccine provided by the invention has higher safety, can quickly induce immune animals to generate high-level neutralizing antibodies, and realizes high-efficiency immune protection of avian adenovirus serum type 4 and serum type 8. Besides, the 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 biotechnology, in particular to a group I serotype 4-serotype 8 avian adenovirus bivalent subunit vaccine and a preparation method thereof. Background technique [0002] Fowl adenovirus (FAV-I) belongs to the family Adenoviridae and belongs to the genus Fowl Adenovirus. Avian adenoviruses are common pathogens of infectious diseases. They are widely present in the respiratory tract and digestive tract of various birds worldwide. Only a few adenoviruses are pathogenic, and most adenoviruses can replicate in birds, but they do not show The clinical symptoms or symptoms are very mild. In the case of mixed infection, adenovirus can become a conditional pathogen, which can cause recessive infection in chicken flocks or cause morbidity and death in chicken flocks as a secondary pathogen. [0003] Avian adenoviruses are divided into three subgroups I, II, and III according to group-specific antigens. Currently, there are ...

Claims

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Application Information

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IPC IPC(8): C07K14/075C12N15/34C12N15/70A61K39/235A61K39/295A61P31/20
Inventor 王增福李跃崔松奇吴宗学史张艳程海波
Owner 乾元浩生物股份有限公司
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