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Replication-defective recombinant human-type-4 adenovirus, and preparation method and application thereof

A replication-deficient, adenovirus technology, applied in the biological field, can solve problems such as high toxicity and side effects, safety, etc.

Pending Publication Date: 2019-12-10
GUANGZHOU N BIOMED LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these strategies either have high toxicity and side effects (such as immunosuppressants), are safe (by modifying or transforming the surface protein of the adenovirus vector), or can only be used once and have limitations (that is, the immune response against the new vector cannot continue to reuse)

Method used

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  • Replication-defective recombinant human-type-4 adenovirus, and preparation method and application thereof
  • Replication-defective recombinant human-type-4 adenovirus, and preparation method and application thereof
  • Replication-defective recombinant human-type-4 adenovirus, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Circularization of the Ad4 adenovirus genome.

[0061] 1. Construction of Ad4 genome circularization shuttle vector.

[0062] The Ad4 genome was used as a template for PCR amplification to obtain recombinant arms Ad4-L and Ad4-R.

[0063] Ad4-L primer sequence:

[0064] Ad4-L Fw, ATAGAATTCGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO. 1);

[0065] Ad4-L RwR, TTTACTAGTGTTTAAACGTAATCGAAACCTCCACGTAATGG (SEQ ID NO. 2).

[0066] PCR program: 95°C, 30 seconds; 62°C, 30 seconds; 72°C, 20 seconds; 25 cycles.

[0067] Ad4-R primer sequence:

[0068] Ad4-R Fw, ACTAGTAGCTGGATCCAAGCCTCGAGGCACTACAATG (SEQ ID NO. 3);

[0069]Ad4-R Rw, CCTGCCGTTCGACGATGCGATCGCCATCATCAATAATATACCTTATAGATGG (SEQ ID NO. 4).

[0070] PCR program: 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 80 seconds; 25 cycles.

[0071] Homologous recombinase (Vazyme) was used to connect to pSIMPLE 19 (EcoRV) vector (TaKaRa) to obtain Ad4 genome circularization shuttle plasmid pT-Ad4(L+R).

[0072] 2. Construction of ...

Embodiment 2

[0074] Example 2: Knockout of E3 gene and construction of pAd55ΔE3 plasmid.

[0075] 1. Construction of E3 gene knockout shuttle plasmid pVax-delE3(L+R).

[0076] The Ad4 genome was used as a template for PCR amplification to obtain recombinant arms L-delE3 and R-delE3.

[0077] L-delE3 primer sequence:

[0078] L-delE3F, GACATTGATTATTGACTAGTTTCAACACCTGGACCACTGCC (SEQ ID NO. 5);

[0079] L-delE3R, ATTTAAATTGGAATTCAAGGTCAGAGACTGGTTGAAGGATG (SEQ ID NO. 6).

[0080] PCR program: 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.

[0081] R-delE3 primer sequence:

[0082] R-delE3F, GAATTCCAATTTAAATAGCAGTCTGGCGATACCAAGG (SEQ ID NO. 7);

[0083] R-delE3R, GTTTAAACGGGCCCCTTAGACATTCTTGGTGGTGACAGGGTC (SEQ ID NO. 8).

[0084] PCR program: 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.

[0085] Homologous recombinase (Vazyme) was used to connect to pVax vector (Invitrogen) to obtain the shuttle plasmid pVax-delE3(L+R) for knocking out the E3 gene. ...

Embodiment 3

[0088] Example 3: Knockout of E1 gene and construction of pAd4ΔE1ΔE3 plasmid.

[0089] 1. Construction of the E1 gene knockout shuttle plasmid pVax-delE3(L+R).

[0090] The Ad4 genome was used as a template for PCR amplification to obtain recombinant arms L-delE1 and R-delE1.

[0091] L-delE1 primer sequence:

[0092] L-delE1F, CCAGATACGCGTGTATACCATCATCAATAATATACCTTATAGATGG (SEQ ID NO. 9);

[0093] L-delE1R, GATATCAAGTTAATTAAAATCGAAACCTCCACGTAAAC (SEQ ID NO. 10). PCR program: 95°C, 30 seconds; 50°C, 30 seconds; 72°C, 20 seconds; 25 cycles.

[0094] R-delE1 primer sequence:

[0095] R-delE1F, TTAATTAACTTGATATCGTGTGGATGTGACGGAGGAC (SEQ ID NO. 11);

[0096] R-delE1R, GCCCAGTAGAAGCGCCGGTGCGGGATTATTAGTGGAACTTGAG (SEQ ID NO. 12).

[0097] PCR program: 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.

[0098] Homologous recombinase (Vazyme) was used to connect to pVax vector (Invitrogen) to obtain the shuttle plasmid pVax-delE1(L+R) for knocking out the E1 gene...

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Abstract

The invention discloses a preparation method and application of replication-defective recombinant human-type-4 adenovirus. The replication-defective recombinant human-type-4 adenovirus is obtained bythe following method that a genome of the human-type-4 adenovirus is made to be like plasmids, genes E1 and E3 of the human-type-4 adenovirus are knocked out, and open reading frames 2, 3, 4, 5 and 6of an gene E4 of Ad4 are replaced with the corresponding open reading frames of an gene E4 of Ad5. The described replication-defective recombinant human-type-4 adenovirus vector can be potentially applied to the research and development of anti human-type-4 adenovirus vaccine, the screening of anti human type 4 adenovirus neutralizing antibody and drugs, the research and development of anti otherpathogen vaccine, report tracer system of biological research and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a replication-deficient recombinant human type 4 adenovirus vector and its preparation method and application. Background technique [0002] Adenovirus (Adenovirus, Ad) is a double-stranded DNA virus with a genome length of about 35-40 kb. It is known that human adenoviruses are divided into 7 subgroups (A-G), including more than 50 serotypes and more than 90 genotypes. After infecting patients, they mainly cause acute respiratory diseases (adenovirus B and C subgroups), conjunctivitis ( Adenovirus subgroups B and D) and gastroenteritis (adenovirus subgroup F 41 and 42, G subgroup 52). Studies have reported that Ad4 outbreaks mainly concentrated in military forces, schools and other places where youths and adolescents gather, and even led to the death of patients. However, there is no specific drug for the treatment of Ad4 infection, and only supportive treatment can be taken clini...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C07K16/08A61K39/235A61P31/20
CPCC12N15/86C07K16/081A61K39/12A61P31/20C12N2710/10343C07K2317/76C12N2710/10334A61K2039/5254
Inventor 陈凌冯立强
Owner GUANGZHOU N BIOMED LTD
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