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Replication-defective adenovirus vector vaccine for co-expressing respiratory syncytial virus pre-fusion protein and adhesion glycoprotein

A technology of fusion glycoprotein and syncytial virus, applied in the direction of virus/phage, virus, vector, etc., can solve the problem of insufficient immunogenicity, and achieve the effect of rapid preparation and strong immune response

Active Publication Date: 2021-01-15
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the genetically engineered vaccines or new vaccines in the prior art rely too much on the neutralizing antigen F protein, neglecting the neutralizing antigen G protein, and there is a problem of insufficient immunogenicity

Method used

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  • Replication-defective adenovirus vector vaccine for co-expressing respiratory syncytial virus pre-fusion protein and adhesion glycoprotein
  • Replication-defective adenovirus vector vaccine for co-expressing respiratory syncytial virus pre-fusion protein and adhesion glycoprotein
  • Replication-defective adenovirus vector vaccine for co-expressing respiratory syncytial virus pre-fusion protein and adhesion glycoprotein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, preF2A3G nucleotide sequence construction

[0059] 1. Design site-directed mutagenesis primers, introduce the three mutation sites N67I, S215P and E487Q into the full-length RSV F gene, phosphorylate the 5' end of the mutation primers, and perform mutation chain synthesis reactions. DpnI digests the amplified products and transforms them into Competent cells, the plasmid was extracted to obtain pcDNA3.1 / TM. See below for site-directed mutagenesis primers:

[0060] N67I primer: 5'-agctgagcaacatcaagaaaatcaagtgcaacggc-3'

[0061] S215P primer: 5'-gcagagctgcagcatccccaacatcgagacagtg-3'

[0062] E487Q primer: 5'-gttccccagcgaccagttcgacgccag-3'

[0063] 2. Design overlapping PCR primers, use pcDNA3.1 / TM as a template, and add GS linker. The post-PCR product was identified by gel electrophoresis to obtain the PreFT fragment, which was cloned into the pMD18-T vector to obtain the plasmid pMD18-T-preFT. Overlapping PCR primers are as follows:

[0064] Upstream...

Embodiment 2

[0074] Example 2. Preparation and in vitro identification of recombinant adenovirus

[0075]1. Digest pShuttle26 / preF2A3G and pShuttle63 obtained in step 2 with restriction endonuclease AflII and SalI respectively, recover the vector fragment and the target fragment by gel electrophoresis, connect, transform and extract the plasmid, and use restriction endonuclease KpnI Digested with XhoI, two bands of about 2800bp and 4000bp were obtained by electrophoresis, and pShuttle63 / preF2A3G was obtained.

[0076] 2. Single-digest pShuttle63 / preF2A3G with restriction endonuclease ScaI, single-digest pChAd63 with restriction endonuclease HpaI, use the method of HiFi DNA Assembly kit, connect the vector fragment and the target fragment, and in DH5α competent Cells were transformed and plasmids were extracted. Single digestion with restriction endonuclease MluI, electrophoresis to obtain two bands of about 7500bp and 28000bp, single digestion with restriction endonuclease NdeI, electroph...

Embodiment 3

[0080] Embodiment 3, the application of recombinant adenovirus

[0081] 1. Animal immunity

[0082] Female BALB / c mice aged 6-8 weeks were divided into 3 groups. On the 0th day, the first intramuscular injection was immunized. On the 21st day after immunization, the blood after the first immunization was collected from the inner canthus vein of the eye. On the 28th day, the second immunization was given by intramuscular injection. On the 49th day, the blood after the second immunization was collected from the inner canthus vein of the eye. On the 56th day, the drug challenge experiment was carried out by nasal drop, and the dose of drug challenge was 1×10 6 pfu / 50 μl of wtRSV.

[0083] The grouping and processing are as follows:

[0084] The first group (G1 group): the second immunization was carried out by intramuscular injection, and the primary immunization was rChAd63 / empty virus solution (the virus amount was 1×10 10 vp), the secondary immune substance is rChAd63 / em...

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Abstract

The invention provides a nucleotide sequence for co-expressing a respiratory syncytial virus pre-fusion glycoprotein (preF) and an adhesion glycoprotein 130-230aa (G130-230) and a preparation method of the nucleotide sequence. The sequence comprises nucleotide sequences of preF and G130-230, wherein the nucleotide sequences of preF and G130-230 are connected together by a T2A sequence; the nucleotide sequence contains a coding sequence shown as a preF2A3G sequence; the nucleotide sequence for coding preF is located at the 5' end of the preF2A3G sequence; the nucleotide sequence for coding G130-230 is located at the 3' end of the preF2A3G sequence; the nucleotide sequence is expressed by a chimpanzee 63-type replication-defective recombinant adenovirus vector or a human 26-type replication-defective recombinant adenovirus vector; the recombinant adenovirus is rChAd63 / preF2A3G or rAd26 / preF2A3G; after the recombinant adenovirus is used for immunizing an animal, a serum antibody aiming atthe fusion glycoprotein and the adhesion glycoprotein can be induced and generated simultaneously; and the induced and generated serum antibody is an antibody aiming at preF mainly and can be appliedto a vaccine for preventing the respiratory syncytial virus. The recombinant adenovirus vaccine can be prepared in a large scale and is used for preventing RSV infection.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a co-expression of respiratory syncytial virus respiratory syncytial virus prefusion glycoprotein (preF) and adhesion glycoprotein 130-230aa (G 130-230 ) nucleotide sequence and its preparation method and carrier. Background technique [0002] Human respiratory syncytial virus (Human respiratory syncytial virus, RSV) is the most important viral pathogen causing lower respiratory tract infection in infants, and almost 100% of infants have been infected more than once within 2 years old. Among the causes of death among children aged 1 month to 1 year, the number of deaths caused by RSV infection accounts for about 6.7% of the total, ranking second after malaria. Among the causes of death of children younger than 5 years old due to lower respiratory tract infection, the number of deaths caused by RSV infection accounted for about 6.29% of the total, ranking second after pneu...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/861C12N7/01A61K39/155A61P31/14
CPCC07K14/005C12N15/86C12N7/00A61K39/12A61P31/14C12N2760/18522C12N2760/18534C12N2710/10021C12N2710/10043C12N2710/10052C07K2319/00C12N2800/22Y02A50/30
Inventor 何金生付远辉彭向雷郑妍鹏
Owner BEIJING JIAOTONG UNIV
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