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Preparation method of avian influenza virus HA gene recombinant adenovirus

An avian influenza virus and recombinant adenovirus technology, applied in the field of vaccines, can solve the problems of inability to express multiple virus subtype antigens, low expression efficiency of HA gene expression system, etc.

Inactive Publication Date: 2015-03-11
TIANJIN RINGPU BIO TECH
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the technical defects of the prior art, and provides a method for preparing a recombinant adenovirus with the HA gene of the avian influenza virus, so as to solve the technical problem of low expression efficiency of the HA gene expression system in the prior art
[0007] Another technical problem solved by the present invention is the technical problem in the prior art that it is impossible to co-express multiple virus subtype antigens with one expression system

Method used

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  • Preparation method of avian influenza virus HA gene recombinant adenovirus
  • Preparation method of avian influenza virus HA gene recombinant adenovirus
  • Preparation method of avian influenza virus HA gene recombinant adenovirus

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Embodiment 1

[0037] Design of primers for target genes and construction of recombinant shuttle vector Primers were designed according to the nucleic acid sequences of H5N1 and H9N2 subtype AIV HA1 provided by the GenBank database, and restriction enzyme sites were introduced on the upstream and downstream of the HA gene. The HA was connected with the linearized pShuttle vector after double digestion with endonucleases Sal I and Xho I, transformed into competent Escherichia coli DH5α, and positive clones were picked for PCR and sequencing identification (sequencing analysis by Shanghai Sangon Company). Primer sequences for identifying positive clones:

[0038] F: 5'-ACGCGTCGACAAAATGAAGGCAATACTAGTGTT-3'

[0039] R: 5'-CGCTCGAGGGGCCCTGGGTTGGACTCGACGTCGCCGGCCAACTTGAG-3'

[0040] PCR reaction cycle conditions: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 30 s, annealing at 60°C for 40 s, extension at 72°C for 50 s (30 cycles); final extension at 72°C for 10 min.

[0041] The i...

Embodiment 2

[0046] For the construction of recombinant adenovirus plasmid and the acquisition of recombinant adenovirus, the recombinant adenovirus shuttle plasmid was linearized with restriction endonuclease Pme I, purified and recovered, and transformed into BJ5183 competent cells containing the adenovirus backbone plasmid pAdeasy-1 for simultaneous Source recombination. Plasmids were extracted after Kan resistance screening, and the extracted plasmids were identified by enzyme digestion and PCR with Pac I. In order to obtain a large number of high-quality recombinant plasmids, the screened positive recombinant adenovirus plasmids were transformed into host bacteria DH5α for amplification.

Embodiment 3

[0048] A method for preparing recombinant adenovirus with HA gene of avian influenza virus, the method comprises the following steps:

[0049] 1) connecting the fragment with the HA gene of the avian influenza virus to the adenovirus shuttle plasmid, introducing the ligation product into Escherichia coli for amplification, and then extracting the recombinant adenovirus shuttle plasmid;

[0050] 2) The recombinant adenovirus shuttle plasmid extracted in step 1) was single digested and then introduced into Escherichia coli containing the adenovirus backbone plasmid for homologous recombination, and then the recombinant adenovirus plasmid was extracted;

[0051] 3) Linearize the recombinant adenovirus plasmid extracted in step 2), transfect it into 293 cells, and package it to obtain the recombinant adenovirus with HA gene of avian influenza virus;

[0052] Simultaneously: the fragment having the HA gene of the avian influenza virus is a fragment of the HA gene of the avian influ...

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Abstract

The invention provides a preparation method of avian influenza virus HA gene recombinant adenovirus, which creatively comprises the following steps: carrying out a series of intermediate processes on plasmid pCAGGS, adenovirus shuttle plasmid pShuttle, adenovirus framework plasmid pAdEasy-1 and the like to obtain a gene expression plasmid and other intermediate products, and transfecting the obtained recombinant adenovirus plasmid with 293 cell; and carrying out immunohistochemical screening on the recombinant virus according to the adenovirus-infected cytopathy and specific cells. By using the CAG as the promoter to express the target gene, the method obviously enhances the expression level of the target gene. The hemagglutinin recombinant adenovirus for respectively expressing H5N1 and H9N2 subtype avian influenza viruses provides a virus model for development of the H5 / H9 subtype avian influenza virus bivalent nucleic acid vaccine, and also lays the foundation for development of the AIV (avian influenza virus) adenovirus live vector vaccine.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to a preparation method of avian influenza virus HA gene recombinant adenovirus. Background technique [0002] Bird flu (Avian influenza) first appeared in Italy in 1878 and was later confirmed to be caused by type A influenza virus. Afterwards, bird flu not only brought huge economic losses to animal husbandry, but also seriously threatened human health and life. At present, H5N1 subtype avian influenza virus has infected humans in many countries. In October 2005, the first confirmed case of human infection with H5N1 subtype avian influenza virus occurred in Anhui Province, my country, and the first human H5N1 subtype avian influenza virus A / Anhui / 1 / 2005 was isolated in my country. December 31, 2011 On the 1st, a person in Shenzhen, my country was infected with a highly pathogenic avian influenza virus and died. Although the Shenzhen Center for Disease Control and Prevention ann...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861
Inventor 王寿山李亚杰郁宏伟杨保收梁武
Owner TIANJIN RINGPU BIO TECH
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