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Replication-defective human adenovirus type 55 vector and preparation method and application thereof

A replication-deficient, adenovirus technology, which is applied in the fields of virus/bacteriophage, botany equipment and methods, biochemical equipment and methods, and can solve problems such as toxic and side effects

Active Publication Date: 2015-08-19
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 relatively high toxic side effects (such as immunosuppressants), or can only be used once and cannot be reused due to the generation of immune responses against new vectors

Method used

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  • Replication-defective human adenovirus type 55 vector and preparation method and application thereof
  • Replication-defective human adenovirus type 55 vector and preparation method and application thereof
  • Replication-defective human adenovirus type 55 vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Knockout of E1 gene and construction of pAd55ΔE1ΔE3-Kana plasmid.

[0066] 1. Construction of the E1 gene knockout shuttle plasmid pVax-delE1(L+R).

[0067] Using the Ad55 genome as a template for PCR amplification, the recombinant arms L-delE1 and R-delE1 were obtained.

[0068] L-delE1 primer sequence:

[0069] L-delE1F, ATAGAATTCGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO. 1);

[0070] L-delE1R, TTTACTAGTGTTTAAACGTAATCGAAACCTCCACGTAATGG (SEQ ID NO. 2).

[0071] PCR program: 95°C, 30 seconds; 62°C, 30 seconds; 72°C, 20 seconds; 25 cycles.

[0072] R-arm primer sequence:

[0073] R-delE1F, ATTTCTAGAGTTTAAACGAGACCGGATCATTTGGTTATTG (SEQ ID NO. 3);

[0074] R-delE1R, AAAGAATTCGGGAAATGCAAATCTGTGAGGG (SEQ ID NO. 4).

[0075] PCR program: 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 80 seconds; 25 cycles.

[0076] L-delE1 was digested with SpeI+EcoRI and ligated to pVax vector (Invitrogen) digested with the same enzyme to obtain pVax-L-delE1; R-delE1 was digested with EcoR...

Embodiment 2

[0079] Example 2: Knockout of Kana resistance gene and construction of pAd55ΔE1ΔE3 plasmid.

[0080] 1. Construction of Kana resistance gene knockout shuttle plasmid pVax-delK(L+R).

[0081] Using the Ad55 genome as a template for PCR amplification, the recombinant arms L-delK and R-delK were obtained.

[0082] L-delK primer sequence:

[0083] L-delK F, ATAACTAGTGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO. 5);

[0084] L-delK R, TTTGAATTCGTTTAAACGTAATCGAAACCTCCACGTAATGG (SEQ ID NO. 6).

[0085] PCR program: 95°C, 30 seconds; 61°C, 30 seconds; 72°C, 20 seconds; 25 cycles.

[0086] R-delK primer sequence:

[0087] R-delK F, ATCGTTTAAACGAGACCGGATCATTTGGTTATTG (SEQ ID NO. 7);

[0088] R-delK R, ATCTCTAGAGGGAAATGCAAATCTGTGAGGG (SEQ ID NO. 8).

[0089] PCR program: 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 80 seconds; 25 cycles.

[0090] L-delK was digested with SpeI+EcoRI and ligated to pVax vector (Invitrogen) digested with the same enzyme to obtain pVax-L-delK; R-delK was digested wi...

Embodiment 3

[0093] Example 3: Transformation of Ad55E4 gene and construction of pAd55ΔE1ΔE3 (5Orf6) and pAd55ΔE1ΔE3 (5E4) plasmids.

[0094] 1. Construction of the shuttle plasmid for genetic modification of Ad55E4.

[0095] 1) Using the Ad55 genome as a template, carry out PCR amplification with the following primers to obtain the Ad55E4 gene.

[0096] R-delE3Mlu, GATCACGCGTGGACTAAGAGACCTGCTACCCATG (SEQ ID NO. 9);

[0097] L-delK R, TGTAGATCTGTTTAAAACCTTTAGCCCCATTACGTCAGTTTAG (SEQ ID NO. 10).

[0098] PCR program: 95°C, 30 seconds; 61°C, 30 seconds; 72°C, 4 minutes; 25 cycles.

[0099] After the end of the PCR product was phosphorylated, it was ligated with a blunt-ended T vector (TaKaRa) to obtain p55E4.

[0100] 2) Using the p55E4 plasmid as a template, PCR amplification was performed to obtain a linearized p55E4 with a SapI restriction site added at the end and the Ad55E4Orf6 gene removed.

[0101] Sap-p55E4R, TTACGCTCTTTCCTAGCCGTGATCCAGACTCCGG (SEQ ID NO. 11);

[0102] Sap-p55E4...

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Abstract

The invention relates to the biological technical field, and particularly discloses a replication-defective human adenovirus type 55 vector and a preparation method and an application thereof. The replication-defective human adenovirus type 55 vector is prepared by the following method: E1 and E3 genes of Ad55 are knocked out, an open reading frame 6 or open reading frames 2, 3, 4, 6 and 6 / 7 of an E4 gene in an Ad55 genome are changed into corresponding reading frames of an Ad5 genome, and in addition, an exogenous gene expression frame is integrated in an E1 gene region of the Ad55. The vector can be produced in large quantities in 293, PerC6 and other auxiliary cell lines, and can be concentrated and purified by density gradient centrifugation; normal human cells do not have the replication capacity, thereby having an attenuated phenotype; in addition, the vector can efficiently express an exogenous gene in target cells. The vector can be used as a vaccine or a gene therapy vector, and can also be applied in development of drugs and neutralizing antibodies and in a trace reporting system.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a replication-deficient human type 55 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 60 serotypes, among which adenovirus types 3, 4, 7, and 14 in subgroup B can cause acute respiratory diseases and even fatal pneumonia after infection. . In recent years, adenovirus type 55 (hereinafter referred to as Ad55), produced by recombination of adenovirus types 11 and 14, has appeared, which has caused a series of community-acquired pneumonia outbreaks and even death cases. However, there is no specific drug and vaccine against Ad55 infection, and only supportive treatment can be adopted clinically. Therefore, the development of anti-Ad55 vaccines and drugs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/64C07K16/08A61K39/235A61K39/12A61K48/00A61P31/20A61P31/14
CPCC12N15/86C12N2710/10321C12N2710/10362C12N2760/14134A61K39/12A61K2039/5256C12N15/861C12N2710/10062C12N2710/10021C12N2710/10034A61K39/0208C12N15/902C12N2710/10334C12N2710/10343C12N2710/10351C12N2710/10371C12N2800/22C12N2800/24
Inventor 陈凌冯立强
Owner GUANGZHOU N BIOMED LTD
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