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Respiratory syncytial virus-like particles and preparation method and application thereof

A technology for syncytial virus and respiratory tract, which is applied to respiratory syncytial virus-like particles and the fields of preparation and application thereof, can solve problems such as low expression level, allergic reaction, etc. Effect

Active Publication Date: 2014-03-19
BEIJING JIAOTONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The research on RSV VLPs vaccine has been reported in the literature. In the past, baculovirus vector system or eukaryotic plasmids were often used to prepare RSV VLPs, but there were obvious shortcomings. Carrying a large number of membrane proteins of insect cells, the VLPs may cause allergic reactions when applied to humans; when RSV VLPs are prepared from eukaryotic plasmids, there is a problem of low expression

Method used

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  • Respiratory syncytial virus-like particles and preparation method and application thereof
  • Respiratory syncytial virus-like particles and preparation method and application thereof
  • Respiratory syncytial virus-like particles and preparation method and application thereof

Examples

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Embodiment 1

[0050] Embodiment 1: Construction of recombinant shuttle plasmid

[0051] 1. Construction: In view of the codon preference of mammalian cells, optimize the codons of wild-type RSV M, F, and G, and name them Msyn, Fsyn, and Gsyn, respectively, to increase their expression in mammalian cells; Whole-gene synthesis of codon-optimized M, F, and G genes, pMA-T-Msyn, pMA-T-Fsyn, and pMA-T-Gsyn; use restriction endonucleases HindⅢ and EcoRⅤ to cut pMA-T-Msyn respectively , pMA-T-Fsyn and pMA-T-Gsyn, and recover the target fragments.

[0052] After agarose gel electrophoresis, near 774bp (see figure 1 ) and 1728bp (see figure 2 ) each has a specific band, which is consistent with the length of Msyn gene and Fsyn gene respectively.

[0053] The shuttle vector pShuttle-CMV was cut with restriction endonucleases HindⅢ and EcoRⅤ, the vector fragments were recovered, and the target fragments Msyn, Fsyn, and Gsyn were ligated overnight at 12°C to 16°C, and the ligation products were name...

Embodiment 2

[0073] Embodiment 2: Construction contains the recombinant adenovirus plasmid of Msyn, Fsyn or Gsyn gene

[0074] 1. In RecA + Homologous recombination in E.coli BJ5183 cells:

[0075] Linearize pShuttle-Msyn, pShuttle-Fsyn, pShuttle-Gsyn with PmeⅠ enzyme digestion, digest overnight at 37°C. Take about 500ng of pShuttle-Msyn DNA molecules, pShuttle-Fsyn DNA molecules, pShuttle-Gsyn DNA molecules after linearization with PmeⅠ, add H 2Add O to 400 μl, add 40 μl 3mol / L sodium acetate (pH 5.2), then add 2-2.5 times the volume of absolute ethanol, and place at -20°C for 30 minutes to precipitate. Centrifuge at 12000rpm for 10min at 4°C. The pellet was washed once with 75% ethanol, dried and dissolved in 10 μl ddH 2 O and stored at -20°C for later use. Take about 5 μl of the pShuttle-Msyn DNA molecules, pShuttle-Fsyn DNA molecules, and pShuttle-Gsyn DNA molecules obtained in the previous step, and chemically transform the RecA containing the adenovirus backbone plasmid pAdEasy-...

Embodiment 3

[0090] Example 3: Transfection of recombinant adenovirus plasmid into 293 cells, cell culture, and obtaining recombinant adenovirus

[0091] 1. Digest recombinant adenoviral plasmids pAdEasy-Fsyn, pAdEasy-Msyn and pAdEasy-Gsyn with Pac I:

[0092] Recombinant adenoviral plasmids pAdEasy-Fsyn, pAdEasy-Msyn, pAdEasy-Gsyn were digested overnight with Pac I, digested completely to remove plasmid components such as ori and Kana resistance genes, and exposed their ITRs, after digestion, precipitated with absolute ethanol, dried, 20 μl sterile HO 2 O dissolved.

[0093] 2. Transfection of 293 cells

[0094] 293 cells were donated by the Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention.

[0095] When the growth abundance of 293 cells is about 50% to 70%, take 10 μl each of the recombined adenovirus plasmids pAdEasy-Fsyn, pAdEasy-Msyn and pAdEasy-Gsyn digested with Pac I enzyme and mix well with 240 μl opti-MEM (GIBCO) ( Solution ...

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Abstract

The invention relates to respiratory syncytial virus-like particles, and establishes a novel method of preparing respiratory syncytial virus-like particles for animal immunization. The preparation method comprises the following steps: according to codon bias of mammalian cells, carrying out codon optimization for M, F and G genes which are respectively cloned to a shuttle vector to obtain recombinant adenovirus plasmids, and transfecting to obtain the recombinant adenovirus FGAd-Msyn, FGAd-Fsyn and FGAd-Gsyn; infecting the recombinant adenovirus by use of Vero cells to obtain the virus-like particles; purifying the virus-like particles by adopting a sucrose density gradient centrifugation method; applying the particles to animal immunization. According to the invention, the virus-like particles are prepared by adopting the adenovirus vector, and a novel virus-like particle preparation method is established. The protein expression quantity is improved and the yield of the virus-like particles is improved through codon optimization, so that a better immune effect is obtained, and the respiratory syncytial virus-like particles are relatively safe.

Description

technical field [0001] The invention relates to respiratory syncytial virus (Human respiratory syncytial virus, RSV) virus-like particles (Virus like particles, VLPs) and a preparation method and application thereof. Background technique [0002] Human respiratory syncytial virus (Human respiratory syncytial virus, RSV) is widely distributed all over the world and is the most important viral pathogen causing lower respiratory tract infection in infants. 6-month-old infants have a high incidence rate, and about 70% of infants are infected within one year of birth. RSV is also the main viral pathogen of acute lower respiratory tract infection in infants and young children in my country, causing epidemic asthmatic pneumonia or bronchiolitis in infants and young children. . The elderly and immunocompromised persons are also susceptible to serious infections caused by RSV. The World Health Organization and others estimate that the annual number of cases worldwide is 64 million ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/45C12N15/861C07K16/10A61K39/155A61P31/14G01N33/569
Inventor 何金生付远辉焦月盈洪涛
Owner BEIJING JIAOTONG UNIV
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