Fowl adenovirus 4 (FAdV-4) vector system and applications thereof

A technology of poultry adenovirus and vector system, applied in the field of recombinant poultry adenovirus FAdV-4 vector system, can solve the problem of viral genome instability and other issues

Active Publication Date: 2018-10-16
中国疾病预防控制中心病毒病预防控制所 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the repeated sequences in the genome of avian adenovirus, homologous recombination in E. coli cells may cause the instability of the virus genome

Method used

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  • Fowl adenovirus 4 (FAdV-4) vector system and applications thereof
  • Fowl adenovirus 4 (FAdV-4) vector system and applications thereof
  • Fowl adenovirus 4 (FAdV-4) vector system and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, FAdV-4 genome is cloned to plasmid

[0051] The schematic diagram of the construction of the backbone plasmid pKFAV4 is shown in figure 1 . Wild-type FAdV-4 was isolated from a broiler chicken farm in Shandong Province, and it can be passaged and amplified in LMH chicken liver cancer cells. Under standard polymerase chain reaction (PCR) conditions, using the pShuttle-CMV plasmid (stratagene company) as a template, 1707KFAdV-F: tgtcaaaaga cgcggttata taagatgat g tttaaac gat cccgagcggt atcag (the underline indicates the PmeI restriction site), 1708KFAdV-R: tgtcaaaagacgcggttata taagatgat gtttaaac tgg aacaacactc aaccctat (the underline indicates the PmeI restriction site) (BGI Synthesis) was used as a primer to amplify the 2537bp fragment KAN-ORI (SEQ ID NO: 1). The 30 bp at both ends of the fragment is identical to the ITR sequence of the FAdV-4 genome. The KAN-ORI fragment recovered by electrophoresis was mixed with wild-type FAdV-4 genomic DNA, and t...

Embodiment 2

[0053] Embodiment 2, construction of intermediate plasmid pKFAV4AP

[0054] The pKFAV4 plasmid is relatively large, and it is difficult to directly modify the genome on the left side of the plasmid. Therefore, a small plasmid is gradually isolated from the pKFAV4 plasmid for exogenous gene replacement.

[0055] figure 2 A schematic diagram of the construction of the intermediate plasmid pKFAV4AP is shown. First digest the pKFAV4 plasmid with AvrII, treat with calf alkaline phosphatase (CIP) to remove 5’p, prevent the fragments from self-ligating, and recover the 16728bp fragment by electrophoresis; then design 2 primers, 1707Avr-PacF: cgaatacgag ttgg cctag ctctcgcaga acagggaatg gggca ttaat taa ccgct (the underline indicates AvrII and PacI restriction sites), 1707Avr-PacR tgtcgtactt cagc cctag ccattggcggagaccgtaag cgg ttaatta a tgcccca (the underline indicates AvrII and PacI restriction sites) self-annealed, and DNA polymerase extended to obtain a 96bp DNA fragment (...

Embodiment 3

[0056] Embodiment 3, construction of shuttle plasmid pKFAV4APNM

[0057] image 3 A schematic diagram of the construction of the shuttle plasmid pKFAV4APNM is shown. The molecular weight of the intermediate plasmid pKFAV4AP is still relatively large, and it is difficult to be directly used for the insertion of the target gene. Therefore, a smaller plasmid was isolated from pKFAV4AP, and Orf1-Orf2 was replaced with human cytomegalovirus promoter-multiple cloning site-SV40polyA plus tail signal to obtain the shuttle plasmid pKFAV4APNM. The specific construction process is as follows: First, the intermediate plasmid pKFAV4AP was digested with NheI, the 6950bp fragment was recovered by electrophoresis, and the pKFAV4APN plasmid was obtained after self-ligation. The pKFAV4APN plasmid was then digested with NheI / AgeI, and a 4942bp fragment was recovered by electrophoresis; the pKFAV4APN plasmid was used as a template and 1707KFAV4AgeIF attcctccac tgctttgaac cca1707KFAV4AgeIR cccgt...

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Abstract

The invention provides a recombinant fowl adenovirus FAdV-4 vector system. The recombinant fowl adenovirus FAdV-4 vector system comprises a framework plasmid pKFAV4, a middle plasmid pKFAV4AP and a shuttle plasmid pKFAV4APNM. A target gene coding region is cloned to a multiple cloning site of the shuttle plasmid, NheI digestion is carried out on the shuttle plasmid carrying a target gene expression cassette, and the obtained fragment replaces the corresponding part of the middle plasmid; AvrII or PacI digestion is carried out on the middle plasmid carrying a target gene, the obtained fragmentreplaces the corresponding part of the framework plasmid by virtue of the ligation reaction or the DNA assembly reaction, and then a recombinant adenovirus plasmid is obtained; a PmeI linearized adenovirus plasmid is used for transfecting LMH cells, and the recombinant FAdV-4 adenovirus realizing control over the expression of the target gene by virtue of a human cytomegalovirus promoter (CMVp) can be prepared. In the prepared recombinant adenovirus, the Orf1-Orf2 regions of the FAdV-4 genome are replaced by the target gene expression cassette, and the other regions of the adenovirus are reserved. The recombinant fowl adenovirus FAdV-4 vector system is expected to have the broad application prospect in the research and development of oral vaccines for birds.

Description

technical field [0001] The invention belongs to the field of recombinant vaccines, and in particular relates to a recombinant adenovirus FAdV-4 vector system and its application. Background technique [0002] The Adenoviridae family is divided into five genera (genus), of which mammalian adenoviruses infect mammals and avian adenoviruses (Aviadenoviruses) infect birds [1] . The genome length of avian adenovirus is 43.7-45.7kb, which is the longest adenovirus genome except white sturgeon adenovirus; its penton contains 2 fibers, encoded by a single gene or 2 genes (1 gene encodes 1 root). Avian adenoviruses include avian adenoviruses, which are classified into five species: A, B, C, D, and E. Fowl adenovirus type 4 (fowl adenovirus 4, FAdV-4) belongs to FAdV-C, and is the pathogen of chicken inclusion body hepatitis-hydropericardium syndrome (HHS). The lethality rate of the disease is 30%-70%, which is seriously harmful to broiler breeding. The research and development o...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66A61K39/235A61P35/00A61P31/16
CPCA61K39/12A61K2039/54A61P31/16A61P35/00C12N15/66C12N15/86C12N2710/10234C12N2710/10243
Inventor 鲁茁壮邹小辉洪涛朱亚露赵阳
Owner 中国疾病预防控制中心病毒病预防控制所
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