Replication defective recombinant adenovirus Ad41 vector system and application thereof

A recombinant adenovirus, replication-deficient technology, applied in the direction of virus/phage, application, and cells modified by introducing foreign genetic material, can solve the problem of reducing the infectivity of Ad5, and achieve the effect of broad application prospects.

Inactive Publication Date: 2010-01-06
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now widely used adenovirus vectors are mostly based on human adenovirus type 5 genome, but gastric acid or intestinal digestive juice can greatly reduce the infectivity of Ad5, so it is not an ideal gene therapy vector for enteral administration (Favier AL, Burmeister WP, Chroboczek J. Uniquephysical properties of human enteric Ad41 responsible for its survival and replication in the gastric testinal tract. Virology, 2004, 322(1): 93-104. [pubmed: 15063120])

Method used

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  • Replication defective recombinant adenovirus Ad41 vector system and application thereof
  • Replication defective recombinant adenovirus Ad41 vector system and application thereof
  • Replication defective recombinant adenovirus Ad41 vector system and application thereof

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Effect test

Embodiment 1

[0023] Embodiment 1, construction of backbone plasmid pAdbone41

[0024] 1) Construction of plasmid pAM-RLarm containing left arm, right arm (L arm, R arm) sequences, replicon and ampicillin resistance gene sequence for recombination

[0025] Under standard polymerase chain reaction (PCR) conditions (50 microliter system: 10 nanograms of template, 30 picomoles of primers, 10 nanomoles of each of the four deoxynucleoside triphosphates, 1 unit of high-fidelity thermostable DNA polymerase ;Reaction conditions: pre-denaturation at 94 degrees Celsius for 2 minutes, denaturation at 94 degrees Celsius for 30 seconds, annealing at a temperature 4 degrees Celsius lower than the melting temperature of the primer for 30 seconds, extension at 68 degrees Celsius, and the extension time is based on the length of the product. Amplify 800 base pairs It takes 1 minute to calculate, 30 cycles of amplification, and 68 degrees Celsius for 5 minutes to fill in the end; the following PCR amplificat...

Embodiment 2

[0029] Embodiment 2, the construction of shuttle plasmid pSh41-CMV ( figure 2 )

[0030] 1) Construction of the cloning plasmid pKan containing the resistance gene (Kan) of kanamycin

[0031] Using pShuttle-CMV (Stratagene Company) as a template, use primer 5F070522: gcccggatcc (BamHI) cgagcggtatcagctcactcaaag (SEQ ID NO: 6) and primer 5R070522: gccccgcgcgc (BssHII) aaactggaacaacactcaaccctat (SEQ ID NO: 7) to amplify the 2483bpt fragment (refer to Schutt - CMV sequence, version017005; fragment position: 4660nt-7122nt), this fragment contains the replication origin and the kanamycin resistance gene. Use endonucleases BamHI and BssHII located at both ends of the fragment to recover after double digestion; primer 5F: cgcgcaaggg gaagagctcg ggaaggggaattcggagaagggtatacc (SEQ ID NO: 8) and primer 5R: gatcggtatacccttctccgaattccccttcccgagctcttccccttg (SEQ ID NO: 9) were mixed and annealed to obtain both ends It is a double-stranded DNA with a length of about 50bp at the cohesive end...

Embodiment 3

[0038] Example 3, construction of packaging cell line 293-E1B

[0039] 1) Cloning of Ad41 E1B55K gene

[0040] The E1B55K gene was amplified by PCR using the DNA of stool samples from Ad41-positive infants with viral diarrhea as a template, and the reaction conditions were the same as before. The primer used is 8F: gccc aagctt (HindIII) atggagcgcccaaacccatctg (SEQ ID NO: 14) and 8R: gccc tctaga (Xbal) tacccttaatcctcatcgctggattc (SEQ ID NO: 15), amplifies a fragment containing the complete CDS of E1B55K. The amplified fragment was connected to the T vector (pMD-18T, TAKARA company), after sequencing confirmed that the sequence was correct (sequencing was carried out at Beijing Aoke Biotechnology Company), it was digested with HindIII and XbaI, and connected to the pcDNA3 plasmid (Invitrogen). Between the restriction sites HindIII and XbaI, the eukaryotic expression plasmid pcDNA3-E1B55K was obtained.

[0041]2) Gene transfection and screening of cell lines

[0042] The p...

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Abstract

The invention relates to a replication defective recombinant adenovirus Ad41 vector system and an application thereof in the field of gene therapy and recombinant vaccines. Specifically, the vector system comprises a backbone plasmid, a shuttle plasmid and a packaging cell line, wherein, the backbone plasmid contains an adenovirus Ad41 genome which deletes an encoding region required for packaging adenovirus, the shuttle plasmid contains multiple cloning sites used for inserting a target gene and an adenovirus Ad41 genomic fragment used for carrying out homologous recombination with the backbone plasmid, and the packaging cell line integrates genes required for packaging adenovirus Ad41 in the cell genome and can stably express the packaging genes. The generated recombinant Ad41 virus has broad application prospects in intestinal gene therapy and research of oral recombinant vaccines.

Description

technical field [0001] The invention relates to a replication-deficient recombinant adenovirus Ad41 vector system and its application in the field of gene therapy and recombinant vaccine. Background technique [0002] The intestinal tract as the target organ of gene therapy has the following advantages: (a) the route of administration is simple, and it can be administered directly orally or endoscopically; (b) the surface area of ​​the intestinal epithelium is large, which is convenient for the sufficient adsorption and infection of the carrier; (c) The crypt cells of the intestinal epithelium are stem cells, and their infection can prolong the expression time of the target gene (Croyle MA, Stone M, Amidon GL, Roessler BJ. In vitro and in vivo assessment of adenovirus 41 as avector for gene delivery to the intestine. Gene Ther, 1998, 5(5): 645-654. [pubmed: 9797869]). The mucous membrane is the first barrier to prevent most pathogenic microorganisms from invading the body. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N5/10C12N7/01
Inventor 鲁茁壮屈建国洪涛
Owner 中国疾病预防控制中心病毒病预防控制所
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