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Zika virus vaccine based on replication-defective recombinant adenovirus vector

A recombinant adenovirus, replication-deficient technology, applied in the field of Zika virus vaccine, can solve the problems of limiting the application of adenovirus vectors, and achieve the effect of improving immunogenicity and expression

Pending Publication Date: 2020-05-01
广州佰芮慷生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a high level of immune response against common human serotypes of adenovirus (Ad5 or Ad2) in the population, which limits the application of adenoviral vectors.

Method used

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  • Zika virus vaccine based on replication-defective recombinant adenovirus vector
  • Zika virus vaccine based on replication-defective recombinant adenovirus vector
  • Zika virus vaccine based on replication-defective recombinant adenovirus vector

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preparation example Construction

[0043] The fourth aspect of the present invention provides a method for preparing the replication-deficient recombinant adenovirus, comprising the following steps:

[0044] (1) Construct the above-mentioned recombinant shuttle plasmid;

[0045] (2) Cloning the recombinant shuttle plasmid into a recombinant adenovirus vector to obtain a replication-deficient recombinant adenovirus vector;

[0046] (3) The replication-deficient recombinant adenovirus vector is linearized and then transfected into virus packaging cells for packaging, amplification and purification to obtain a replication-defective recombinant adenovirus.

[0047] In the present invention, the specific type of the virus packaging cells is not particularly limited, and the virus packaging cells well-known to those skilled in the art can be used. Preferably, the virus packaging cells are HEK 293 cells, but not limited thereto.

[0048] Wherein, the endonuclease to be linearized in step (3) is Pac I.

[0049] In th...

Embodiment 1

[0066] Example 1. Acquisition and optimization of JE signal peptide and exogenous genes prM and E to improve the expression of exogenous genes.

[0067] The previously reported amino acid sequence of the signal peptide of Japanese encephalitis virus (JEV) is MGKRSAGSIMWLASLAVVIACAGA, which is coded according to the Zika virus sequence, using codons that have appeared in the signal peptide or Zika virus sequence, and finally Determine the nucleotide sequence of the JE signal peptide as shown in SEQ ID NO:1. The gene sequences of prM and E of Zika virus are derived from the Asian Zika virus strain Z16006 (GenBankno.KU955589.1), and the codons are optimized using the software Upgene, making foreign genes more suitable for expression in mammalian cells. The nucleotide sequence of optimized prM-E is shown in SEQ ID NO:2. The determined gene sequence was synthesized by BGI to obtain the JE signal peptide and the optimized exogenous gene sequence.

Embodiment 2

[0068] Example 2. Construction of the recombinant shuttle plasmid pShuttle2-CMV-JE-prM-E.

[0069] The nucleotide sequence containing the JE signal peptide synthesized by the whole gene in Example 1, and the plasmid encoding the nucleotide sequence JE-prM-E of the membrane protein prM and envelope glycoprotein E of Zika virus were used EcoRI and BamHI Carry out double enzyme digestion, recover the enzyme digestion product, connect the enzyme digestion product with the shuttle plasmid pShuttle2-CMV-Flag double digestion with EcoRI and BamHI, transform, and plate to obtain the recombinant shuttle plasmid pShuttle2-CMV-JE-prM- e.

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Abstract

The invention discloses a Zika virus vaccine based on a replication-defective recombinant adenovirus vector. A nucleotide sequence of a JE signal peptide, and an optimized nucleotide sequence encodingZika virus membrane protein prM and envelope glycoprotein E are cloned into a shuttle plasmid vector pShutle2-CMV-Flag to obtain a recombinant shuttle plasmid, so that the expression of foreign proteins is significantly increased, and at the same time immunogenicity of an antigen is improved. Meanwhile, a recombinant adenovirus expression vector SAd23-L is used to escape pre-existing immune responses against common adenovirus vectors, and the humoral and cellular immunity with a higher level can be induced to generate in animals. After the recombinant adenovirus as a Zika virus vaccine is used to immunize animals, the humoral and cellular response to Zika virus is quickly induced to generate, especially a neutralizing antibody with a high level is induced to generate, and the specific cellular response to Zika virus antigen M and E protein is induced to generate in mice. Therefore, the Zika virus vaccine can be used to prevent large-scale outbreak and epidemic of Zika virus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Zika virus vaccine based on a replication defective recombinant adenovirus vector. Background technique [0002] Zika virus (Zika virus, ZIKV) was discovered in rhesus monkeys in the Zika jungle in Uganda in 1947. It is a single-stranded positive-sense RNA virus belonging to the Flaviviridae family. According to the statistics of the World Health Organization, more than 30 countries and regions in the Americas and Africa have reported the spread of Zika virus. Zika virus, which poses little threat to the general population, can cause mild fever or skin rashes, and some people may also experience conjunctivitis, muscle and joint pain, and fatigue. However, infection by pregnant women can interfere with the neurodevelopmental system of the fetus, which may lead to miscarriage, microcephaly or even death. At the same time, Zika virus is also a trigger factor of Guillain Barre syndro...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/861C12N15/66A61K39/12A61P31/14
CPCC12N15/63C12N15/86C12N15/66A61K39/12A61P31/14C12N2710/10043C12N2770/24134A61K2039/5256A61K2039/575Y02A50/30
Inventor 罗升学刘博超张攀丽
Owner 广州佰芮慷生物科技有限公司
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