Replicating type recombinant adenovirus HAdV-5 carrier system and application thereof

A technology of recombinant adenovirus and vector system, which is applied in the field of replicative recombinant adenovirus HAdV-5 vector system, can solve the problem that the virus cannot replicate, and achieve the effect of wide application

Active Publication Date: 2018-05-18
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

[0004] There are currently a variety of commercial HAdV-5-based replication-deficient vector systems, however, the virus cannot replicate after this vector infects target cells and only functions as a gene transfer vector
There is no report on commercial replicating HAdV-5 vector system

Method used

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  • Replicating type recombinant adenovirus HAdV-5 carrier system and application thereof
  • Replicating type recombinant adenovirus HAdV-5 carrier system and application thereof
  • Replicating type recombinant adenovirus HAdV-5 carrier system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, construction of backbone plasmid pKAd5

[0054] The schematic diagram of the construction of the backbone plasmid pKAd5 is shown in figure 1 . Under standard polymerase chain reaction (PCR) conditions, using the pShuttle-CMV plasmid (purchased from Stratagene) as a template, 1710KAd5F: caatccaaaa taaggtatattattgatgat g ttaattaa g atcccgagcg gtatcag (the underline indicates the PacI restriction site), 1710KAd5R: aatccaaaat aaggtatatt attgatgatg ttaattaa tg gaacaacact caaccctat (the underline indicates the PacI restriction site) (the above primer was synthesized by BGI) was used as primer to amplify the 2540bp fragment ORI-KAN (SEQ ID NO: 2). About 30 bp at both ends of this fragment are identical to the ITR sequence of HAdV-5 genome. Mix the ORI-KAN fragment recovered by electrophoresis with wild-type HAdV-5 (VR-1516, purchased from ATCC) genomic DNA (see Genbank accession number: AY339865 for the sequence), and then add an equal volume of DNA assembly...

Embodiment 2

[0056] Embodiment 2, construction of intermediate plasmid pMDXE3YA

[0057] In order to facilitate the transformation of the adenovirus E3 region, the NdeI fragment containing the E3 region was isolated from the backbone plasmid pKAd5 constructed in Example 1, and E3 was replaced with the YFP coding region and the SV40 polyA signal region, and the plasmid obtained after the replacement was named is pMDXE3YA.

[0058] figure 2 A schematic diagram of the construction of the intermediate plasmid pMDXE3GA is shown.

[0059] The construction process of the intermediate plasmid pMDXE3GA is as follows:

[0060] 1) NdeI / EcoRI double digestion of the backbone plasmid pKAd5 constructed in Example 1, electrophoresis recovery of 7776 bp fragment (NdeI-EcoRI, the sequence can be found in SEQ ID NO: 3, SEQ ID NO: 3 is due to retaining the complete NdeI site and EcoRI site point, actually 7783bp).

[0061] 2) Using the pMD-18T plasmid as a template (purchased from Takara Company), using...

Embodiment 3

[0064] Embodiment 3, construction of shuttle plasmid pKNXE3M

[0065] The target gene replacement operation on the intermediate plasmid pMDXE3YA involves multi-step PCR and DNA assembly, which is cumbersome. In order to further simplify the cloning of the target gene, the target gene region of the E3 region was isolated again from the intermediate plasmid pMDXE3YA, and a multiple cloning site was added to construct a smaller shuttle plasmid pKNXE3M. image 3 A schematic diagram of the construction of the shuttle plasmid pKNXE3M is shown.

[0066] The construction method of the shuttle plasmid pKNXE3M is as follows: with pmCherry-N1 plasmid (purchased from Clontech Company) as template, with 1712KNEOf:ggcc gaattc cacttttcgg ggaaatgtgc (the underline indicates the EcoRI restriction site) and 1712KNEOr: ggcc gaattc cgcaggaaag aacatgtgag (the underline indicates the EcoRI restriction site) is used as a primer to amplify the 2600bp fragment, after electrophoresis recovery, it...

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Abstract

The invention provides a replicating type recombinant adenovirus HAdV-5 carrier system. The system comprises a framework plasmid pKAd5, an intermediate plasmid pMDXE3YA and a shuttle plasmid pKNXE3M;a target gene coding region is cloned to multiple cloning sites of the shuttle plasmid, EcoRI digests the shuttle plasmid carrying a target gene, and an obtained fragment is used for replacing the corresponding part of the intermediate plasmid; then NdeI is used for digesting the newly-constructed intermediate plasmid, and an obtained fragment is used for replacing the corresponding part of the framework plasmid so as to obtain a recombinant adenovirus plasmid; and the PacI linearized recombinant adenovirus plasmid is used to transfect a 293 cell so as to prepare a recombinant HAdV-5 adenovirus with target gene expression controlled by an HAdV-5 major later promoter (MLP). according to the system, the recombinant adenovirus can be replicated and proliferated in various human cells, is a replicating type virus and is expected to have a wide application prospect in the research and development of animal oral carrier vaccines.

Description

technical field [0001] The invention belongs to the field of recombinant vaccines, in particular, the invention relates to a replicative recombinant adenovirus HAdV-5 vector system and its application. Background technique [0002] Oral vaccines have many advantages [1，2] . Intramuscular injection of vaccines requires the participation of medical staff and multiple immunizations, which requires a large amount of vaccines, and the purification process is relatively complicated and costly. Oral vaccines can make up for the shortage of intramuscular vaccines. The dose of live vaccines is small, no strict purification is required, and the cost is low; the vaccination is convenient, no professional medical staff is required, and the compliance is good. The mucous membrane is the entry barrier for pathogens, and oral vaccines can trigger strong mucosal immunity, directly intercepting pathogens outside the body; attenuated oral vaccines can trigger strong cellular immunity, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861
CPCC12N15/86C12N2710/10043
Inventor 鲁茁壮刘红军邹小辉
Owner 中国疾病预防控制中心病毒病预防控制所
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