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Neutralizing epitope of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and application thereof

An antigenic epitope, adenovirus technology, applied in the direction of application, antiviral agent, antiviral immunoglobulin, etc., can solve the problem of inability to prevent other types of adenovirus infection, adenovirus vaccine strain infection, and inability to effectively resist 3 adenovirus infection

Inactive Publication Date: 2011-01-12
STATE KEY LAB OF RESPIRATORY DISEASE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the neutralizing antibody produced by adenovirus can only resist the infection of the same type of adenovirus and cannot prevent the infection of other types of adenovirus, the antibodies produced by the adenovirus type 4 and 7 vaccines cannot effectively resist the infection of adenovirus type 3
In addition, the type 4 and 7 adenovirus vaccines used abroad are oral live virus vaccines. The vaccine can produce asymptomatic infection in the intestinal tract to achieve the effect of immunization, but the adenovirus vaccine strains excreted through feces still have the possibility of infection
Moreover, due to the homology of genes between different subtypes of adenoviruses, homologous recombination may occur between two or more different subtypes of adenovirus vaccines administered at the same time, resulting in new, infectious adenovirus particles, bringing new risk of infection

Method used

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  • Neutralizing epitope of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and application thereof
  • Neutralizing epitope of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and application thereof
  • Neutralizing epitope of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Bioinformatics analysis predicts hypervariable regions (HypervariableRegions, HVRs) of human adenovirus

[0021] The specific steps of the bioinformatics analysis are as follows: firstly, the amino acid sequence of the human adenovirus hexon protein was compared, and then the possible neutralizing antigen epitope was predicted by using the "Antigenic" tool in Emboss. Then, the 3D three-dimensional structure of the human adenovirus type 3, 7 hexon was modeled and the potential neutralizing epitope on the 3D structure was mapped. Finally, the sites exposed on the surface of the hexon "loop" were found and localized to non-constant regions (hypervariable regions) on the sequence.

[0022] figure 1 It is a three-dimensional diagram of the above analysis results, row A is a top view, and row B is a side view. by right figure 1 By analyzing the results, the potential neutralizing epitope on the human adenovirus type 3, 7 hexon can be located and its amino acid s...

Embodiment 2

[0033] Embodiment 2: construct recombinant virus

[0034] In order to analyze the predicted neutralizing epitopes of the hypervariable region (HVR) of type 3 and type 7 human adenoviruses through serum neutralization test and challenge test, the inventors used overlapping PCR (overlapping PCR) method to construct recombinant virus.

[0035] The HVR1, 2, 5 and 7 sequences of type 7 adenovirus were replaced on the PBR322-L-R plasmid by overlapping PCR (overlapping PCR), and then homologously recombined with the PBR-Adv3-EGFP plasmid. The specifically obtained recombinant plasmids are PBR-Adv3(Adv7-1)-EGFP, PBR-Adv3(Adv7-2)-EGFP, PBR-Adv3(Adv7-5)-EGFP, PBR-Adv3(Adv7-7)-EGFP, Among them, PBR-Adv3(Adv7-1)-EGFP means that hypervariable region 1 of Adv3 is replaced by hypervariable region 1 of Adv7, and so on.

[0036] Transfect cells with the positive plasmids obtained from the above recombination to obtain recombinant viruses MH1, MH2, MH5 and MH7; wherein recombinant virus MH1 is...

Embodiment 3

[0037] Embodiment 3: Serum neutralization test

[0038]To prepare antiserum, a serum neutralization test was performed. The purified recombinant viruses MH1, MH2, MH5 and MH7, wild type 3 ADV virus (ADV3E) and wild type 7 ADV virus (ADV7) with enhanced green fluorescent protein (EGFP) were injected intraperitoneally into Balb / C mice, respectively. The polyclonal antibodies of MH1, MH2, MH5 and MH7 as well as ADV3E and ADV7 were obtained respectively, and then the neutralization test was carried out. The neutralization test was divided into two groups: the first group was to detect the neutralization reaction between the anti-ADV3E and ADV7 serum and the recombinant virus MH1, MH2, MH5 and MH7, in order to detect whether the neutralizing antigen expression of ADV3 and ADV7 was included in the recombinant virus The second group is to detect the neutralization reaction between the antisera of MH1, MH2, MH5, MH7, ADV3E, ADV7 and normal control mice and ADV3, to detect whether the...

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Abstract

The invention discloses three neutralizing epitope of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and an application thereof. The amino acid sequence of the neutralizing epitope of the HAdV-3 and HAdV-7 is selected from the amino acid sequence shown in SEQ ID NO: 1, 2, 3, and the nucleotide sequence of the neutralizing epitope of HAdV-3 and HAdV-7 is selected from the nucleotide sequence shown in the SEQ ID NO: 4, 5, 6. The neutralizing epitopes of HAdV-3 and HAdV-7 can be used for preparing vaccines or antibody and antigen binding fragments for preventing infection of human adenovirus type 3 and human adenovirus type 7, and the antibody and the antigen binding fragments can be used for preparing drugs for preventing or curing adenovirus infection. The drugs contain the antibody or antigen binding fragments. The invention also provides a method for preventing and curing adenovirus infection, i.e. the vaccines or drugs with immune effective quantity are applied. The three neutralized epitopes of HAdV-3 and HAdV-7 can be served as target protein for developing a diagnostic kit.

Description

technical field [0001] The invention belongs to the technical field of biomedical engineering, and specifically relates to neutralizing antigenic epitopes of human type 3 and type 7 adenoviruses and applications thereof. Background technique [0002] The adenovirus particle is a symmetrical structure of an icosahedron, and the shell is composed of 252 shell subunits, among which the 12 pre-angles of the icosahedron are penton protein and a fiber protrusion with a length of 10-30nm; The other 240 non-vertical particles are composed of hexon trimers, which can stimulate the body to produce neutralizing antibodies and group and type-specific antibodies. The amino acid sequences of the hexon proteins of different types of adenoviruses have as high as 78-95% homology, and the changes are mainly concentrated in the hypervariable regions (HVRs) exposed on the surface of the virus capsid; The recognition sites of neutralizing antibodies are concentrated on these hypervariable regio...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K14/075C07K16/08C12N15/34A61K39/235A61K39/395A61P31/20G01N33/569
Inventor 周荣邱红玲周志超李潇苏晓波高文娟钟南山
Owner STATE KEY LAB OF RESPIRATORY DISEASE
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