Type B acian metapneumovirus subunit vaccine
An avian metapneumovirus and subunit vaccine technology, which can be applied in vaccines, viruses, antiviral agents, etc., can solve the problem of high safety, and achieve the effects of good safety, improved antibody level, and improved uniformity
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Embodiment 1
[0021] Example 1: Amplification and sequence analysis of F protein
[0022] 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.
[0023] 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.
[0024] 1. Amplification of Type B Avian Metapneumovirus SHS / A3 Strain ...
Embodiment 2
[0035] Embodiment 2: Recombinant expression of F protein
[0036] 1. The preparation method of recombinant type B avian metapneumovirus F protein
[0037] 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.
[0038] a. Construct expression vector:
[0039] 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 linea...
Embodiment 3
[0044] Embodiment 3: the preparation of subunit vaccine
[0045] 1. Subunit vaccine preparation
[0046] 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 pack 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
[0047] 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 minute...
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