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Adenoviral vector system and recombinant adenovirus construction method

A technology of recombinant adenovirus and vector system, applied in the field of gene therapy, can solve the problems of long time and complicated construction steps, and achieve the effect of wide application

Active Publication Date: 2019-02-01
中国疾病预防控制中心病毒病预防控制所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires the purchase of a linearized pAdenoX vector, the foreign gene insertion site and other parts of the recombinant virus genome have been determined, and cannot be used for artificial transformation of recombinant adenovirus
[0005] The existing adenovirus vector systems all have defects, some construction steps are cumbersome and time-consuming, some introduce unnecessary foreign sequences into the recombinant virus, and some cannot be used for reverse genetic transformation of recombinant adenovirus

Method used

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  • Adenoviral vector system and recombinant adenovirus construction method
  • Adenoviral vector system and recombinant adenovirus construction method
  • Adenoviral vector system and recombinant adenovirus construction method

Examples

Experimental program
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Effect test

Embodiment 1

[0063] Example 1, construction of adenoviral plasmid pAd5f11p-EF1a carrying fusion fiber gene (F5-11p) and human EF1a promoter

[0064] Using the backbone plasmid pAdEasyf11p carrying the F5-11p fusion gene to construct an adenovirus plasmid containing the human EF1a promoter in the E1 region, see figure 1 .

[0065] First replace the CMV promoter of the pShuttle-CMV plasmid with the human EF1a promoter. Using the pShuttle plasmid as a template, and using 1411Sh5EF1aF1: ccggtgtaca caggaagtga caat and 1411Sh5EF1aR1: cttttgtatgaattactcga cgtcagtatt acgcgctatg agtaacacaa as primers, a 181bp product was amplified by PCR, which started from the upstream BsrGI site and contained the HAdV-5 packaging signal ( 再以pLVX-EF1a-Tet3G质粒为模板,1411Sh5EF1aF2: cgcgtaatac tgacgtcgag taattcatac aaaaggactc gc和1411Sh5EF1aR2:acggtacctc acgacacctg aaatggaaga a为引物,PCR扩增得到1360bp产物,该产物含有人EF1a启动子;再将1411Sh5EF1aF3:ttccatttca ggtgtcgtga ggtaccgtcg acgcggccgcacgcgttcta and 1411Sh5EF1aR3: ggccgatatc ttagctagca...

Embodiment 2

[0066] Example 2, removing the original PmeI site of the pAd5f11p-EF1a adenovirus plasmid

[0067] For the construction process of integrating the F5-11p fusion gene and the mutated PmeI site (mutated from gtttaaac to gtttaaat) into the same adenovirus plasmid, see figure 2 .

[0068] In the previously constructed adenovirus plasmid pAd5GXP [10] , the original PmeI site (gtttaaac) of the HAdV-5 genome has been artificially mutated into gtttaaat, which is a synonymous mutation and does not affect the amino acid sequence encoded by the viral gene containing this site. The original PmeI site of the HAdV-5 genome was removed by mutation, and the purpose was to reserve the site for foreign gene cloning. Digest pAd5f11p-EF1a plasmid with BamHI, recover the 10999bp fragment containing the F5-11p gene; digest pAd5GXP with BamHI, treat with calf alkaline phosphatase (CIP), remove 5'P, and recover the 24594bp fragment by electrophoresis; the above two fragments are connected and iden...

Embodiment 3

[0069] Example 3. Introducing a PmeI site at the cloning site of the exogenous gene in the E1 region of the adenovirus

[0070] For further modification of the cloning site of the exogenous gene in the E1 region of the adenovirus, see image 3 . In order to facilitate molecular biological operations such as site-directed mutagenesis, a smaller plasmid was first isolated from the pAd5f11p-EF1a adenovirus plasmid constructed in Example 1.

[0071] The pAd5f11p-EF1a plasmid was first digested with BstZ17I, and the 7210bp fragment containing the cloning site of the foreign gene was recovered by electrophoresis. In order to restore the modified fragment in the later stage, two oligonucleotide primers were designed and synthesized, namely 1805F11p-EF1PmeA1: gctgtccgtg tccccgtata cagacttgag aggcctgtcg aattcccaaaataaggta and 1805F11p-EF1PmeA2: gccgcatagt taagccagta tacggatcct taattaacatcatcaataact gattc atacgactt After extension, a 109bp double-stranded DNA product was obtained; the...

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Abstract

The invention provides an adenoviral vector system and a recombinant adenovirus construction method. The vector system comprises adenoviral plasmids pKAd5f11p-EF1aP and pKAd5f11pES-PmeI and shuttle plasmids pUC19-PM. The adenoviral plasmid contains an E1 / E3 deleted human adenovirus type 5 (HAdV-5) genome and the original HAdV-5 fiber gene is replaced by a fusion gene F5-11p of HAdV-5 and HAdV-11p.A PmeI site is an exogenous gene insertion site. Plasmids pKAd5f11p-EF1aP contain a human EF1a promoter in the original E1 region. The shuttle plasmids pUC19-PM are matched with the plasmids pKAd5f11pES-PmeI. The recombinant adenovirus construction method comprises: carrying out PCR amplification to obtain a desired gene fragment containing homologous overlapping regions on both sides and carrying out DNA assembling on the desired gene fragment and PmeI-linearized adenoviral plasmids to obtain adenovirus plasmids containing the desired exogenous gene, or cloning the multiple gene fragments tothe shuttle plasmids, shearing all the desired gene fragments through a restriction endonuclease and carrying out DNA assembling on the desired gene fragments and PmeI linearized pKAd5f11pES-PmeI toobtain the adenovirus plasmids containing the desired exogenous gene.

Description

technical field [0001] The invention belongs to the field of gene therapy, in particular, the invention relates to a set of adenovirus vector system and a method for constructing recombinant adenovirus. Background technique [0002] Adenovirus is a linear double-stranded DNA virus without a cell membrane, and the family Adenoviridae is currently divided into 5 genera [1] Many types of adenoviruses have been developed as gene transfer vectors, which are widely used in basic scientific research, gene therapy and vector vaccines [2-4] . [0003] The genome of adenovirus is about 26-46kb long, which makes the construction of adenovirus vectors different from common plasmid vectors (generally less than 10kb in length). Too long genomes make it very difficult to enrich a multiple cloning site on an adenoviral vector. Several methods have been developed for the construction of recombinant adenoviruses [5-7] . [0004] The construction of adenovirus vectors has gone through fou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/70A61K48/00A61K39/235A61P31/20
CPCA61K39/12A61K48/00A61P31/20C12N15/70C12N15/86C12N2710/10034C12N2710/10043
Inventor 刘红岩鲁茁壮洪涛
Owner 中国疾病预防控制中心病毒病预防控制所
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