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IIIV vaccine containing recombination virus and its united immune preparation

A technology for immune preparations and recombinant adenoviruses, applied in antiviral agents, biochemical equipment and methods, applications, etc., can solve the problems of virus genetic background and activity influence, adenovirus genome prone to mutation, etc.

Inactive Publication Date: 2010-07-07
曾毅
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In terms of adenovirus construction technology, the pAdEasy system designed by He et al. is widely used now. The success rate of this system is only 18-34%, and the virus genome recombination is completed in prokaryotic cells. In theory, the survival rate is lost. Stressed adenovirus genomes are more prone to mutations, and the genetic background and activity of the virus may be affected

Method used

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  • IIIV vaccine containing recombination virus and its united immune preparation
  • IIIV vaccine containing recombination virus and its united immune preparation
  • IIIV vaccine containing recombination virus and its united immune preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Preparation of the transformed gag gene (mod.gag) of embodiment 1

[0065] In this example, through codon optimization, a modified gag gene (mod.gag) capable of expressing in eukaryotic cells independent of the regulatory protein Rev was obtained.

[0066] 1.1 Cloning of HIV-1 gag gene and acquisition of consensus sequence

[0067] Collect 20 samples of venous blood from HIV-1 antibody-positive patients in HIV-1 endemic areas in Henan, 5ml each, anticoagulate with heparin, use Qiagen’s QiaAmp Blood reagent, extract cellular DNA according to the instructions, and store nucleic acid samples at -20°C. The gag gene was amplified by nested-PCR method. Use G-1 / G-2 (G-1: 5'CGA CGC AGG ACT CGG CTT GC 3'; G-2: 5'CCT GGCTTT AAT TTT AC 3') as the outer primers for the first PCR reaction, The conditions are: pre-denaturation at 94°C for 5 min, 30 cycles at 94°C for 45 sec, 55°C for 45 sec, and 72°C for 210 sec; 72°C for 10 min. Take one tenth of the PCR product and use G-3 / G-4 (...

Embodiment 2

[0071] Construction and identification of the recombinant virus of embodiment 2 containing mod.gag gene

[0072] 2.1 Construction and passage of replication-deficient recombinant adenovirus containing mod.gag gene

[0073] 2.1.1 Construction of recombinant adenovirus

[0074] Using the plasmid pUC57-mod.gag as a template, primers hn-a / hn-2 (hn-a: 5'CGG AAT TCT ACC GCC ACCAT 3'; hn-2: 5'GCG TCG ACT TAT TGT GAC GAG GGG T 3') Perform PCR reaction for the upstream and downstream primers respectively, the conditions are: 94°C 5min pre-denaturation; 94°C 45sec, 55°C 45sec, 72°C 105sec, 30 cycles; 72°C 10min. The PCR product was separated by 1% agarose gel electrophoresis, and the 1.5 kb size-specific amplification band was recovered after being judged to be correct with the control. The recovered PCR product and the shuttle plasmid pDC316 (purchased from Canada Microbix Biosystems, Inc. company), respectively recovered the former 1.5kb mod.gag gene fragment and the latter 3.9kb pD...

Embodiment 3

[0093] Example 3 Recombinant virus immune effect test

[0094] 3.1 Mouse immunization experiment of recombinant adenovirus

[0095] Forty female BALB / c mice, 4-6 weeks old, weighing about 18-25 grams, were randomly divided into 8 groups, 5 mice in each group. 4 groups were used to detect the immune effect of rAd5-mod.gag in mice, and the other 4 groups were used to detect the immune effect of rAd5 / F35-mod.gag in mice. The two recombinant viruses used the same dose. Individual immunizations were performed with different doses of rAd5-mod.gag or rAd5 / F35-mod.gag vaccines. Dilute the two recombinant virus vaccines to 10 7 PFU / ml, 10 8 PFU / ml and 10 9 PFU / ml, unilateral tibialis anterior muscle injection, 100 μl each time, and the control group was injected with the same volume of sterile PBS. See Table 1 for specific immunization procedures and doses.

[0096] Table 1. Individual immunization schedules of different doses of recombinant adenovirus vaccines

[0097] ...

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Abstract

An HIV vaccine containing composite virus for preventing and treating AIDS and / or HIV infection is prepared through the codon optimization to the nucleotide sequence of the amplified HIV gag gene, and using the modified gag gene (mod.gag) as the vaccine gene to configure and obtain the recombinant viruses rAd5-mod.gag, rAd5 / F35-mod.gag and rAAV / -mod.gag, which have high cellular and humoral immunoresponses. A combined immune vaccine and its scheme are also disclosed.

Description

field of invention [0001] The invention belongs to the fields of bioengineering and AIDS prevention and treatment. In particular, the present invention relates to vaccines capable of preventing and / or treating AIDS and / or HIV infection. More specifically, the invention provides a recombinant virus vector vaccine containing the gag gene of the modified HIV-1 epidemic strain in China and its combined immunization preparation. Background of the invention [0002] HIV, the human immunodeficiency virus (HIV), is a member of the family Retroviridae, the genus Lentivirus, and the group Primate Lentivirus. Acquired Immunodeficiency Syndrome (AIDS) caused by its infection to humans is the largest infectious disease that directly threatens human health in the world. Since the HIV epidemic, it is estimated that more than 60 million people have been infected and more than 20 million people have died from HIV / AIDS. According to WHO statistics, as of December 2004, there were 39.4 mill...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P31/18C12N15/861
Inventor 曾毅余双庆冯霞刘新蕾刘红梅
Owner 曾毅
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