Fowl type-4 adenovirus vector for target infection on mammalian cells and application of fowl type-4 adenovirus vector

A mammalian and adenovirus technology, applied in the directions of virus/phage, whole cell/virus/DNA/RNA components, application, etc., can solve the problems of low infection efficiency, lack of efficient mammalian FAdV vector, lack of target cells, etc. To achieve the effect of improving infection efficiency

Active Publication Date: 2020-06-02
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

The reason for the low infection efficiency is generally due to the lack of corresponding receptors for fiber in target cells
[0006] At present, there is a lack of high-efficiency FAdV vectors that can be applied to mam

Method used

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  • Fowl type-4 adenovirus vector for target infection on mammalian cells and application of fowl type-4 adenovirus vector
  • Fowl type-4 adenovirus vector for target infection on mammalian cells and application of fowl type-4 adenovirus vector
  • Fowl type-4 adenovirus vector for target infection on mammalian cells and application of fowl type-4 adenovirus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, construct the shuttle plasmid pMD-FAV4Fs carrying avian type 4 adenovirus (FAdV-4) fiber gene

[0070] For the construction process of the shuttle plasmid pMD-FAV4Fs carrying the fiber gene of avian adenovirus type 4 (FAdV-4), see figure 1 .

[0071] 以pKFAV4GFP质粒为模板,1805FAV4FSC1:gccagtgccg gtacctttcg gagggcgacg和1805FAV4FSC2:gaccatgatt acgatatcct cctttggacc catggtagt为引物PCR扩增2999bp;以pMD-18T质粒为模板,1805FAV4FSC3:ggtccaaagg aggatatcgt aatcatggtc atagctgtttcctgt和1805FAV4FSC4:cctccgaaag gtaccggcac tggccgtcgt tttac为引物PCR扩 Increased by 2662bp; the above two bands were recovered by electrophoresis, and the DNA assembly reaction (NEBuilder HiFi DNA Assembly MasterMix) was carried out. The assembly product was transformed into Escherichia coli TOP10 competent strain, spread on LB plate containing ampicillin, and obtained plasmid pMD-FAV4Fs by screening.

[0072] The plasmid contains all and a small amount of sequences between the FAdV-4 genome MauBI / SbfI restriction si...

Embodiment 2

[0073] Example 2, inserting the coding sequence of the RGD4C short peptide into the Knob coding region of the pMD-FAV4Fs shuttle plasmid fiber1 gene

[0074] RGD4C is a short peptide (CDCRGDCFC) containing 9 amino acid residues, and tgt gac tgc cgc gga gactgt ttc tgc is selected as its coding sequence. Insert the RGD4C coding sequence into the CD loop coding region of the pMD-FAV4Fs shuttle plasmid fiber1 gene to obtain the construction process of pMD-FAV4F1CDR as follows figure 2 shown.

[0075] 以pMD-FAV4Fs质粒为模板,1805FAV4F1Mf:gtccataggc tattacatct acatggtg和1805FAV4F1CDRr:agaaacagtc tccgcggcag tcacagctcg cgcctgtgag gtc为引物PCR扩增片段,长度为119bp,以1805FAV4F1CDRf:tgactgccgc ggagactgtt tctgcggaga aaacagcctgactagcg和1805FAV4F1Mr:accctgatag gaaaaaggga tagga为引物PCR扩增 The fragment is 475bp in length. SacI / Agel double enzyme digestion pMD-FAV4Fs, the length of the product is 483 and 5124bp, and the 5124bp fragment is recovered by electrophoresis. The above 119bp, 475bp and 5124bp were subject...

Embodiment 3

[0079] Embodiment 3, introducing genetically modified fiber1 gene into FAdV-4 adenoviral plasmid

[0080] The fiber gene in the shuttle plasmid pMD-FAV4F1CDR was introduced into pKFAV4GFP to obtain the adenovirus plasmid pKFAV4F1CDR-GFP. The construction process is as follows: image 3 shown.

[0081] The pMD-FAV4F1CDR obtained in Example 2 was digested with KpnI / EcoRV, and a 2997bp fragment was recovered after electrophoresis; pKFAV4GFP (2903, 43078bp) was digested with MauBI / SbfI, and a 43078bp fragment was recovered after electrophoresis; 43078bp and 2969bp were subjected to DNA assembly, The adenoviral plasmid pKFAV4F1CDR-GFP was obtained.

[0082] By replacing pMD-FAV4F1CDR with pMD-FAV4F1DER, pMD-FAV4F1HIR or pMD-FAV4F1IJR, the pKFAV4F1DER-GFP, pKFAV4F1HIR-GFP or pKFAV4F1IJR-GFP adenoviral plasmids can be obtained respectively through the above operations.

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Abstract

The invention discloses a fowl type-4 adenovirus vector for target infection on mammalian cells and application of the fowl type-4 adenovirus vector. The vector comprises a genome sequence of a fowl type-4 adenovirus, a replication origin nucleotide sequence of pBR322 and a kanamycin resistance nucleotide sequence, wherein a fiber nucleotide sequence in the genome sequence is artificially transformed, including inserting an RGD4C short peptide nucleotide sequence into a Knob region of a fiber gene, and/or the Knob region of the fiber gene is replaced by a nucleotide sequence of human type-35 adenovirus fiber Knob. The transformed fowl type-4 adenovirus vector disclosed by the invention is capable of improving the infection efficiency on the mammalian cells, and can be adopted to prepare recombinant vaccines or gene treatment kits of mammals.

Description

technical field [0001] The invention belongs to the field of gene therapy and recombinant vaccines, in particular, the invention relates to an avian type 4 adenovirus vector targeted to infect mammalian cells and its application. Background technique [0002] Adenovirus vectors have the following characteristics: large foreign gene load, high expression efficiency; infecting many types of cells, can infect both dividing cells and non-dividing cells; easy to prepare, amplify and purify; non-enveloped virus, Stable physical and chemical properties, high viability, and long storage period; after infecting cells, it exists in the form of episomes outside the chromosome, and does not integrate into the host cell chromosome, so it is safe. Therefore, adenoviral vectors have been widely used in the fields of gene therapy and recombinant vaccines. [0003] Adenoviridae includes 5 genera including mammalian adenovirus and avian adenovirus. At present, most commonly used adenovirus v...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66A61K48/00A61K39/00
CPCC12N15/86C12N15/66A61K48/0008A61K39/00C12N2710/10043A61K2039/53
Inventor 鲁茁壮郭小娟颜丙玉邹小辉
Owner 中国疾病预防控制中心病毒病预防控制所
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