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B-type avian metapneumovirus F protein

A technology of avian metapneumovirus and protein, which is applied in the direction of viruses, virus peptides, antiviral agents, etc., can solve problems such as interference with epidemic monitoring, virulence reversion, and impact on the protection effect of virus attack, and achieve convenient and accurate detection methods and stable antigens Highly specific and highly specific effects

Active Publication Date: 2016-06-22
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional inactivated vaccines and attenuated vaccines have unique advantages such as uniform antibodies after immunization and high antibody titers. However, the antigenicity of the strains used to manufacture vaccines and the clinically popular strains is quite different, which affects the The protective effect against viruses restricts the application of inactivated vaccines
Another problem with attenuated vaccines is that the virulence may return to strong. There are cases of disease after vaccination, and biological safety cannot be fully guaranteed.
Moreover, antibodies produced after immunization with traditional vaccines prepared with whole virus antigens cannot be clinically distinguished from wild virus infection or vaccine immunization, which interferes with the monitoring of the epidemic

Method used

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  • B-type avian metapneumovirus F protein
  • B-type avian metapneumovirus F protein
  • B-type avian metapneumovirus F protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Amplification and sequence analysis of F protein

[0019] In 2010, symptoms of swollen head syndrome appeared in several breeding chicken farms in Shandong Province, and the affected chickens had been injected with the existing avian pneumovirus vaccine before, and it was speculated that the infected virus had mutated; Screening for lung viruses. Finally, an avian pneumovirus SHS / A3 was screened out.

[0020] In order to verify the antigenicity of the screened virus, virus strains from 5 different sources, including the screened strain SHS / A3, were used as antigens to prepare vaccines. After immunizing SPF chickens, the SHS / A3 strain virus solution was used to challenge the virus. The results It shows that compared with other poultry pneumovirus vaccines; the vaccine prepared by itself has a better immune effect (p<0.05); therefore, it is determined that genetic variation has occurred.

[0021] 1. Amplification of Type B Avian Metapneumovirus SHS / A3 Strain ...

Embodiment 2

[0032] Embodiment 2: Recombinant expression of F protein

[0033] 1. The preparation method of recombinant type B avian metapneumovirus F protein

[0034] The method comprises the following steps: a. constructing an expression vector; b. constructing an expression strain; c. inducing, extracting and purifying the recombinant F protein.

[0035] a. Construct expression vector:

[0036] The positive clone plasmid pMD18-T-F and the expression vector pPICZα vector were double-digested with KpnI and NotI, respectively, electrophoresed on a 1.2% agarose gel, and then recovered with a DNA gel recovery kit to obtain about 1.6kb and 3.3kb fragments, respectively. The pPICZα-F expression vector was constructed by directional ligation at 16°C ( Figure 4 It is the enzyme digestion identification diagram of the high-efficiency expression vector of the embodiment of the present invention); after sequencing and verifying that the sequence and reading frame are correct, the plasmid is line...

Embodiment 3

[0041] Embodiment 3: the preparation of subunit vaccine

[0042] 1. Subunit vaccine preparation

[0043] 1. Preparation of bacterial solution for making seedlings Inoculate X33-F strains in YPD liquid medium containing bleomycin, and shake and culture at 30°C for 16-18 hours. Then streak and inoculate in YPD solid medium with bleomycin, select 2 to 3 typical colonies and mix them in a small amount of YPD liquid medium, place them in a shaker at 30°C for 18 hours, and quantitatively aliquot them. After inspection, it is used as a first-class seed. Take the first-grade seeds and inoculate them in BMGY liquid medium, culture them with shaking at 30°C for 16-18 hours, and store them at 2-8°C after microscopic examination

[0044]2. Preparation of protein for seedling production. Add BMGY incomplete liquid medium according to 60% (V / V) of the volume of the fermenter, and add antifoaming agent according to 0.1% (V / V) of the medium, and pass through high-temperature steam for 30 mi...

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Abstract

The invention provides a B-type avian metapneumovirus F protein. The amino acid sequence of the B-type avian metapneumovirus F protein is SEQ ID NO: 1. The F protein can be used for preparing vaccine. B-type avian metapneumovirus subunit vaccine prepared by the F protein has the features that antigen stability, purity and specificity are high, other uncorrelated antibodies are not generated, and a detection method is convenient and accurate. The B-type avian metapneumovirus F protein lays a firm foundation for industrial production of the B-type avian metapneumovirus subunit vaccine.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, in particular to a type B avian metapneumovirus F protein. Background technique [0002] Avian metapneumovirus belongs to the paramyxovirus pneumovirus genus, and its genome is a single-stranded negative-sense RNA without segments, which can cause respiratory diseases in turkeys, called turkey rhinotracheitis; after infecting chickens, it can cause viral air sacculitis, Rhinotracheitis and swollen head syndrome, decreased egg production. In the late 1970s, the disease caused by avian metapneumovirus was first reported in South Africa, and subsequently occurred in North America, South America, the Middle East, and the Far East. The virus was first isolated in the United States in 1989. While isolating the virus, it is often possible to isolate Bordetella, Pasteurella, Pseudomonas, Rhinotracheal Avian bacillus, and Alcaligenes faecalis. Avian metapneumovirus can inhibit the activity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/115C12N15/45A61K39/155A61P31/14
CPCA61K39/12C07K14/005A61K2039/54C12N2760/18334C12N2760/18322
Inventor 刘新文范根成胡潇宫晓李陆梅刘蕾
Owner YEBIO BIOENG OF QINGDAO
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