Respiratory syncytial virus full-length pre-fusion glycoprotein nucleotide sequence, recombinant adenovirus vector and application product thereof

A technology for fusing glycoprotein and nucleotide sequence, which is applied in the fields of fusion glycoprotein nucleotide sequence before full-length fusion of respiratory syncytial virus, recombinant adenovirus vector and its application products, and can solve the problem of low human infection rate and the like

Active Publication Date: 2021-01-15
BEIJING JIAOTONG UNIV
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  • Description
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Problems solved by technology

In view of the advantages of chimpanzee adenovirus serotype 63 (ChAd63) and human adenovirus 26 (human adenovirus serotype 26, Ad26) in humans with low infection rates and high immunogenicity, ChAd63 has not been used as an RSV vaccine Reason for development

Method used

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  • Respiratory syncytial virus full-length pre-fusion glycoprotein nucleotide sequence, recombinant adenovirus vector and application product thereof
  • Respiratory syncytial virus full-length pre-fusion glycoprotein nucleotide sequence, recombinant adenovirus vector and application product thereof
  • Respiratory syncytial virus full-length pre-fusion glycoprotein nucleotide sequence, recombinant adenovirus vector and application product thereof

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preparation example Construction

[0043] The present invention provides a preparation method of recombinant adenovirus and a product containing the recombinant adenovirus, the active ingredient of which is the recombinant adenovirus obtained above, and the product can be a respiratory syncytial virus vaccine and be used for neutralizing respiratory syncytial virus Virus medicines, etc., for example, can be chimpanzee type 63 replication-deficient adenovirus vector vaccine carrying the prefusion glycoprotein of respiratory syncytial virus.

[0044] The preparation method of the recombinant adenovirus provided by the present invention comprises the steps of transfecting the recombinant adenovirus plasmid into the packaging cells, and then culturing the cells to obtain the adenovirus packaging cells, the adenovirus packaging cells have type 5 adenovirus E1 gene. The recombinant plasmid is obtained by inserting the specific DNA molecule into the orangutan type 63 replication defective adenovirus vector; the specifi...

Embodiment 1

[0045] Embodiment 1, the preparation of recombinant adenovirus

[0046] 1. Construction of shuttle plasmids (pShuttle63 and pShuttle26)

[0047] The artificially synthesized DNA sequence was inserted between the two Pac I restriction sites of the pShuttle-CMV vector, transformed into DH5α competent cells, and the plasmids were extracted to obtain the shuttle plasmids pShuttle63 and pShuttle26.

[0048] 2. Construction of backbone plasmids (pChAd63 and pAd26)

[0049] The E1 and E3 regions of the deleted adenovirus were obtained by restriction enzyme ligation and DNA Assembly methods, and E4orf6 was replaced by the backbone plasmids pChAd63 and pAd26 of human type 5 adenovirus E4orf6.

[0050] 3. Construction of shuttle plasmids carrying exogenous genes

[0051] 1. Design mDS-Cav1 site-directed mutagenesis primers, introduce the four mutation sites S155C, S190F, V207L ​​and S290C into the full-length RSV F gene, phosphorylate the 5' end of the mutation primers, and carry out ...

Embodiment 2

[0070] Embodiment 2, the application of recombinant adenovirus

[0071] 1. Animal immunity

[0072] Female BALB / c mice aged 6-8 weeks were divided into 4 groups, 5 mice in each group, and were treated as follows:

[0073] The first group (G1 group): a single immunization was carried out by nasal drops, and the immune substance was rChAd63-empty virus solution (the virus amount was 1×10 10 VP);

[0074] The second group (G2 group): a single immunization was carried out by nasal drop method, and the immune substance was rChAd63-Fsyn virus liquid (the virus amount was 1×10 10 VP);

[0075] The third group (G3 group): a single immunization was carried out by nasal drops, and the immune substance was rChAd63-mDS-Cav1 virus solution (the virus amount was 1×10 10 VP);

[0076] The fourth group (G4 group): a single immunization was carried out by intramuscular injection, and the immune substance was FI-RSV virus liquid (the virus amount was 1.25×10 6 PFU);

[0077] The fifth gr...

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Abstract

The invention provides a respiratory syncytial virus full-length pre-fusion glycoprotein nucleotide sequence, a recombinant adenovirus vector and an application product thereof. The full-length pre-fusion glycoprotein nucleotide sequence comprises four mutation loci including S155C, S190F, V207L and S290C, a transmembrane region and an intracellular region of fusion glycoprotein are reserved, anda sequence table of the full-length pre-fusion glycoprotein nucleotide sequence is shown as a sequence 1. The invention further provides a chimpanzee replication-defective recombinant adenovirus vector pChAd63 or a human replication-defective recombinant adenovirus vector pAd26 containing the full-length pre-fusion glycoprotein nucleotide sequence, an initial strain of the adenovirus vector is chimpanzee adenovirus 63 type or human adenovirus 26 type, adenovirus E1 and E3 regions are deleted, and E4orf6 is replaced with human 5-type adenovirus E4orf6. The invention further provides a product applying the recombinant adenovirus. After the recombinant adenovirus vector is used for immunizing animals, the bodies can be induced to generate strong cellular immunity and humoral immunity reactions in a short time, and the generated antibody has high recognition capability on pre-fusion F and has a good immune effect and an immune protection effect.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a full-length pre-fusion glycoprotein nucleotide sequence of respiratory syncytial virus, a recombinant adenovirus vector and its application products. Background technique [0002] Human respiratory syncytial virus (RSV) is an important viral pathogen that causes lower respiratory tract infections in infants and young children, and is also an important viral pathogen that causes hospitalization and death in high-risk groups such as immunocompromised patients and the elderly. From the perspective of infant infection, 70% of infants have been infected with RSV at the age of 1, and all of them have been infected at least once at the age of 2. From the perspective of the death of children infected with RSV, among the causes of death among children aged 1 month to 1 year, the number of deaths caused by RSV infection accounted for about 6.7% of the total, ranking second after m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12N15/861A61K39/155A61P31/14
CPCC07K14/005C12N15/86A61K39/12A61P31/14C12N2760/18522C12N2710/10343C12N2760/18534A61K2039/575A61K2039/57Y02A50/30
Inventor 何金生付远辉刘美琴彭向雷郑妍鹏黄蕾
Owner BEIJING JIAOTONG UNIV
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