Replicative recombinant human 55-type Adenovirus vector and preparation method and application thereof

An adenovirus, replicating technology, applied in the field of replicating recombinant human 55 adenovirus vector and its preparation, can solve the problems of refractory screening of recombinant clones, high-efficiency acquisition, and genome plasmids not being effectively cut, etc., to improve effect of effect

Active Publication Date: 2016-01-27
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this technology is that partial enzyme digestion often leads to the ineffective cutting of

Method used

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  • Replicative recombinant human 55-type Adenovirus vector and preparation method and application thereof
  • Replicative recombinant human 55-type Adenovirus vector and preparation method and application thereof
  • Replicative recombinant human 55-type Adenovirus vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Circularization of Ad55 genome and construction of pAd55 genome plasmid.

[0061] 1. Construction of the circularized shuttle plasmid.

[0062] Using the Ad55 genome as a template, PCR amplification was performed with the following primers to obtain the recombinant arms L-arm and R-arm.

[0063] L-arm primer sequence:

[0064] L-armF, AAGCTTGCGATCGCCATCATCAATAATATACCTTATAGATGG; 1 (SEQ ID NO. 1) L-armR, TCTAGATGATCTGAATTCAGTATTTCGTTTCCAGTCTCAGC (SEQ ID NO. 2).

[0065] L-arm amplification program: 95°C, 5 minutes; 95°C, 30 seconds; 57°C, 30 seconds; 72°C, 40 seconds; 25 cycles; 72°C, 5 minutes; 12°C, ∞. The amplified product is 700bp.

[0066] R-arm primer sequence:

[0067] R-armF, ATGGAATTCACAGATGGATCCAGCATGAATAATTCTTGA (SEQ ID NO. 3);

[0068] R-armR, ATTTCTAGAGCGATCGCCATCATCAATAATATACCTTATAGAT (SEQ ID NO. 4).

[0069] R-arm amplification program: 95°C, 5 minutes; 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 40 seconds; 25 cycles; 72°C, 5 minutes; 12°C, ...

Embodiment 2

[0074] Example 2: Knockout of Ad55E3 gene and construction of pAd55-E3-Kana plasmid.

[0075] 1. Construction of E3 gene knockout shuttle plasmid.

[0076] Using the Ad55 genome as a template, PCR amplification was performed with the following primers to obtain the upstream and downstream homologous recombination arms L-delE3 and R-delE3 of the E3 region.

[0077] L-delE3 primer sequence:

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

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

[0080] L-delE3 amplification program: 95°C, 5 minutes; 95°C, 30 seconds; 63°C, 30 seconds; 72°C, 30 seconds; 25 cycles; 72°C, 5 minutes; 12°C, ∞. The amplification product is 500bp.

[0081] R-delE3 primer sequence:

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

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

[0084] R-delE3 amplification program: 95°C, 5 minutes; 95°C, 30 seconds; 61°C, 30...

Embodiment 3

[0088] Example 3: Knockout of kana resistance gene and construction of pAd55ΔE3 plasmid.

[0089] 1. Construction of a shuttle plasmid for Kana resistance gene knockout.

[0090] Using the Ad55 genome as a template, PCR amplification was performed with the following primers to obtain the upstream and downstream homologous recombination arms L-delK and R-delK of the E3 region.

[0091] L-delK primer sequence:

[0092] L-delKF, GGTACTAGTTTTGTGGAGGAGTTTTACTCCCTCTG (SEQ ID NO. 9);

[0093] L-delKR, GATGAATTCATTTAAATAGGAAAAGTTTCGTTCTTCTGGTTG (SEQ ID NO. 10).

[0094] L-delK amplification program: 95°C, 5 minutes; 95°C, 30 seconds; 63°C, 30 seconds; 72°C, 30 seconds; 25 cycles; 72°C, 5 minutes; 12°C, ∞. The amplification product is 500bp.

[0095] R-delK primer sequence:

[0096] R-delKF, GATATCATTTAAATGGACTAAGAGACCTGCTACCCATG (SEQ ID NO. 11);

[0097] R-delKR, TGTTCTAGACAGTCTTAAACAAGTGGTGTGGTGG (SEQ ID NO. 12).

[0098] R-delK amplification program: 95°C, 5 minutes; 95°C, 30...

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Abstract

The invention discloses a replicative recombinant human 55-type Adenovirus vector and a preparation method and application thereof. A human 55-type Adenovirus genome is cyclized through homologous recombination technology, E3 genes of Ad55 are knocked out through homologous recombination and double resistance screening technology, and an exogenous gene expression cassette can be integrated in; after Ad55 vector plasmids deleted in the E3 genes are linearized, successful rescue and mass production can be realized by transfecting mammalian cells; further, a purified recombinant Ad55 vector is obtained through density gradient centrifugation, efficient expression in target cells can be realized by using the vector to carry exogenous genes, and activity of exogenous reporter genes can reflect growth characteristics of virus. The replicative recombinant vector based on human Ad55 can be potentially applied to research and development of vaccines resistant to human 55-type Adenovirus, screening of drug and neutralizing antibodies resistant to the human 55-type Adenovirus, research and development of vaccines resistant to other pathogens and development of report tracer systems for biological study.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a replication-type recombinant human type 55 adenovirus vector and its preparation method and application. Background technique [0002] Adenovirus is a non-enveloped double-stranded DNA virus with a genome of about 35-40kb. It is known that there are 7 subgroups (A-G) and more than 60 serotypes of human adenovirus, among which types 3, 4, 7, and 14 infect Acute respiratory illness and even fatal pneumonia can result. In recent years, adenovirus type 55 (hereinafter referred to as Ad55), produced by the recombination of human adenovirus type 11 and type 14, has appeared. Because the population generally lacks immunity against this virus, it is easy to cause respiratory infectious diseases, especially in the military. Groups living in closed environments such as recruits, boarding schools, and childcare institutions. At present, there is no specific treatment drug for ad...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66A61K48/00A61K39/235A61P31/20C07K16/08
Inventor 陈凌冯立强孙彩军秦成峰
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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