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Two recombinant adenoviruses for expressing gE protein of varicella-zoster virus and application

A technology of herpes zoster virus and recombinant adenovirus, applied in the field of biomedicine, can solve problems such as high cost

Pending Publication Date: 2022-03-11
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In June 2020, the vaccine (Xin'an Lishi) was launched in China, and although it is in short supply in the domestic market, its high cost reminds us of the urgent need to develop a shingles vaccine with independent intellectual property rights

Method used

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  • Two recombinant adenoviruses for expressing gE protein of varicella-zoster virus and application
  • Two recombinant adenoviruses for expressing gE protein of varicella-zoster virus and application
  • Two recombinant adenoviruses for expressing gE protein of varicella-zoster virus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Construction and Identification of Recombinant Adenovirus Plasmid

[0047] 1. Construction of the shuttle plasmid

[0048] Use restriction endonucleases SalI and KpnI to double-enzyme pShuttle26 and the artificially synthesized gE nucleotide sequence respectively, recover the carrier fragment and the target fragment by gel electrophoresis, connect and transform the competent bacteria DH10B, and obtain pShuttle26 / gE.

[0049] Restriction endonucleases SalI and KpnI were used to double-digest pShuttle63 and the artificially synthesized gE nucleotide sequence, respectively, and the carrier fragment and the target fragment were recovered by gel electrophoresis, connected and transformed into competent bacteria DH10B to obtain pShuttle63 / gE.

[0050] 2. Construction of recombinant adenovirus plasmid

[0051] Digest pShuttle26 / gE with restriction endonuclease BamHI and MluI, recover the target fragment by gel electrophoresis, and single-digest pAd26 with restriction endonucl...

Embodiment 2

[0054] Rescue and expression identification of recombinant adenovirus

[0055] The recombinant adenoviral plasmid pAd26 / gE was digested with restriction endonucleases PacI and SpeI, and recovered by ethanol precipitation. The obtained linearized recombinant adenoviral DNA genome was transfected into 293 cells with Lipofectamine 2000, and the recombinant adenovirus rAd26 / gE expressing herpes zoster gE protein was rescued; the recombinant adenoviral plasmid pChAd63 was digested with restriction endonuclease PacI / gE, recovered by ethanol precipitation. The obtained linearized recombinant adenovirus DNA genomes were transfected into 293 cells with Lipofectamine 2000, and the recombinant adenovirus rChAd63 / gE expressing herpes zoster gE protein was rescued.

[0056] Take 2 μl each of the purified recombinant adenoviruses rAd26 / gE and rChAd63 / gE, inoculate them in 293 cells respectively, collect the cells after 80% of the cells have lesions, and use the gE protein monoclonal antib...

Embodiment 3

[0058] Shingles vaccine prime-boost

[0059] 1. Animal immunity

[0060] Female C57BL / 6 mice aged 6-8 weeks were divided into 5 groups, on the 0th day, the basic serum was collected, on the 1st day, the first intramuscular injection was immunized; on the 21st day after immunization, the second immunization was injected intramuscularly, twice intramuscular injection The dose was the same (Shingrix immunization dose was 5 μg / 50 μl / mouse, rAd26 / gE and rChAd63 / gE immunization dose was 10 8 PFU / 100 μl / mouse). On the 28th day, the rats were sacrificed to collect splenic lymphocytes and post-immunization serum.

[0061] The grouping and processing are as follows:

[0062] The first group (G1 group): negative control (PBS priming-PBS strengthening group);

[0063] The second group (G2 group): positive control (Shingrix priming-Shingrix boosting group);

[0064] The third group (G3 group): rAd26 / gE priming-rAd26 / gE boosting group;

[0065] The fourth group (G4 group): rChAd63 / gE ...

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Abstract

The invention provides two recombinant adenoviruses for expressing gE protein of varicella-zoster virus and application, and relates to the technical field of biological medicine. The invention provides two recombinant adenoviruses prepared on the basis of the nucleotide sequence of the varicella-zoster virus gE protein, and the recombinant adenoviruses capable of expressing the varicella-zoster virus gE protein are homologous or heterologous combined as the herpes zoster virus vaccine. The immunopotentiator can induce the body to generate strong cellular and humoral immune response in a short time on a mouse.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to two recombinant adenoviruses expressing gE protein of varicella-zoster virus and their application. Background technique [0002] Humans are the only natural host of varicella-zoster virus (Varicella-Zoster Virus, VZV), and its infection targets are mainly children. Almost all children have been infected with VZV during their growth, and children are infected with After recovery, a small amount of virus will remain dormant in the dorsal root ganglia of the spinal cord or the sensory ganglia of the cranial nerves for a long time. The virus in the node will replicate in large numbers and spread along the sensory nerve to the skin tissue of the innervated area to replicate, causing nerve damage and herpes zoster (Herpes zoster). Herpes zoster is mainly seen in middle-aged and elderly people. As the host's cellular immunity against VZV decreases with age, the incidenc...

Claims

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Application Information

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IPC IPC(8): C07K14/04C12N15/38C12N7/01C12N15/861A61K39/25A61P31/22
CPCC07K14/005C12N7/00C12N15/86A61K39/12A61P31/22C12N2710/16722C12N2710/16734C12N2710/10021C12N2710/10043C12N2800/22C12N2800/107A61K2039/5256A61K2039/575A61K2039/57A61K2039/543A61K2039/54A61K2039/541
Inventor 付远辉何金生黄蕾虞结梅彭向雷郑妍鹏
Owner BEIJING JIAOTONG UNIV
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