Recombinant adenovirus vector expressed in-vivo self-assembly respiratory syncytial virus-like particle, preparation method and application thereof

A technology of recombinant adenovirus and syncytial virus, applied in the field of genetic engineering, can solve the problems of reducing immunogenicity, restriction, high cost of expression and purification, etc., and achieve the effects of improving safety, increasing expression amount, and simple preparation

Inactive Publication Date: 2017-08-08
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, the in vitro preparation, expression and purification of VLPs are expensive, and the purification process may also cause protein conformational changes and reduce immunogenicity
These deficiencies have brought great limitations to its clinical application

Method used

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  • Recombinant adenovirus vector expressed in-vivo self-assembly respiratory syncytial virus-like particle, preparation method and application thereof
  • Recombinant adenovirus vector expressed in-vivo self-assembly respiratory syncytial virus-like particle, preparation method and application thereof
  • Recombinant adenovirus vector expressed in-vivo self-assembly respiratory syncytial virus-like particle, preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1 recombination shuttle plasmid

[0045] 1. Construction: In order to increase the expression of RSV F and M proteins in mammalian cells, the codons of RSV F and M were optimized using the codons preferred by mammalian cells and the whole gene was synthesized. The PCR method used 2A sequences to connect F and M proteins. M, after double digestion with Xho I and Sac II, the target fragment was connected to the pShuttle vector.

[0046] 2. Enzyme digestion identification and sequencing: The obtained plasmid was identified by Xho I and Sac II double enzyme digestion, and agarose gel electrophoresis results showed specific bands at 2547bp and 7500bp ( figure 1 ), which were consistent with the size of F-M and pShuttle vectors, respectively, and the plasmids with positive results identified by enzyme digestion were verified to be correct plasmids by sequencing, and the recombinant plasmid pShuttle-F-M was successfully constructed.

Embodiment 2

[0047] Embodiment 2 constructs the recombinant adenovirus plasmid containing F-M gene

[0048] 1. Linearized shuttle plasmid and recombination

[0049] The recombinant plasmid pShuttle-F-M obtained by Pme I linearization, 0.5 μg of the linearized pShuttle-F-M was transformed into the competent cells of BJ5183 carrying the adenovirus genome pAdeasy-1 for recombination, Kana + The resistant LB plate was cultured at 37°C for 16 hours, a single colony was picked for culture, and the plasmid was extracted.

[0050] 2. Enzyme screening and identification of recombinant plasmids

[0051] The obtained plasmid was digested with Pac I, and the recombinant plasmid digestion result was detected by agarose gel electrophoresis ( figure 2 ), electrophoresis results showed that two fragments of 4.5Kb and 33Kb were obtained. The correct recombinant plasmid pAdeasy-F-M was obtained by enzyme digestion.

[0052] 3. Amplification of recombinant plasmids

[0053] The obtained correct recombi...

Embodiment 3

[0054] Example 3 Transfection of HEK293 cells to package recombinant adenovirus

[0055] 1. Pac I linearized recombinant adenovirus plasmid pAdeasy-F-M

[0056] Digest the recombinant adenovirus plasmid pAdeasy-F-M with Pac I, precipitate with ethanol, dry at room temperature, and dissolve in 20 μl of sterilized deionized water.

[0057] 2. Transfect HEK293 cells

[0058] HEK293 cells were obtained from the Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention.

[0059] HEK293 cells were inoculated into cell culture plates (6-well plates), and the cell abundance was about 50%-70% during transfection; take 10 μl Pac I linearized recombinant adenovirus plasmid pAdeasy-F-M, add 240 μl opti-MEM (Gibco) and mix Mix well, add 10 μl Lipofactamine 2000 to 240 μl opti-MEM and mix, incubate at room temperature for 5 min; mix the above two solutions, incubate at room temperature for 20 min. The above 500 μl transfection complex was added ...

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Abstract

The invention discloses a recombinant adenovirus expression self-assembly respiratory syncytial virus-like particle. The virus-like particle comprises respiratory syncytial virus F and M protein, the F and M protein are encoded by optimized F gene and M gene, the F gene and M gene are connected by a 2A sequence, and a nucleotide sequence obtained after connection is shown as SEQ ID No.4. The invention also discloses a preparation method of the respiratory syncytial virus-like particle and application in preparation of vaccines or drugs for prevention or treatment of respiratory syncytial virus infection, respiratory syncytial virus antibodies and diagnostic reagents for respiratory syncytial virus. The invention adopts single recombinant adenovirus as the vector, and establishes a method for in-vivo self-assembly expression of respiratory syncytial virus-like particle, recombinant adenovirus preparation is relatively simple, the cost is remarkably reduce, good immune effect and immune protection are acquired, and security is provided.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, it relates to a recombinant adenovirus vector expressing in vivo self-assembled respiratory syncytial virus-like particles and its preparation method and application. Background technique [0002] Human respiratory syncytial virus (Human respiratory syncytial virus, RSV) is the most important viral pathogen causing lower respiratory tract infection in infants under 5 years old worldwide, often causing pneumonia and bronchitis (Hall, C.B., E.A. Simoes, and L.J.Anderson, Clinical and epidemiologic features of respiratory syncytial virus. Curr Top Microbiol Immunol, 2013.372: p.39-57.). The elderly and immunocompromised patients are also susceptible to RSV (Jorquera, P.A., L.Anderson, and R.A. Tripp, Understanding respiratory syncytial virus (RSV) vaccine development and aspects of disease pathogenesis. Expert Rev Vaccines, 2016.15(2): p. 173-87.). People can be repeatedly in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/861C07K16/10A61K39/155A61P31/14
CPCA61K39/12A61K2039/5258A61K2039/543C07K16/1027C12N7/00C12N15/861C12N2710/10043C12N2760/18523C12N2760/18534
Inventor 何金生彭向雷华颖付远辉
Owner BEIJING JIAOTONG UNIV
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