Construction method of recombinant baculovirus for expressing serum type 4 avian adenovirus fibroid protein F2

A technology of recombinant baculovirus and avian adenovirus, applied in the direction of virus, virus/phage, double-stranded DNA virus, etc., can solve the problem of lack of rapid and specific detection of FAdV-4, and achieve complete post-translational processing and modification ability, expression The effect of high level and high expression

Inactive Publication Date: 2020-08-14
YANGZHOU UNIV
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Problems solved by technology

As there is no serological method for rapid and spec

Method used

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  • Construction method of recombinant baculovirus for expressing serum type 4 avian adenovirus fibroid protein F2
  • Construction method of recombinant baculovirus for expressing serum type 4 avian adenovirus fibroid protein F2
  • Construction method of recombinant baculovirus for expressing serum type 4 avian adenovirus fibroid protein F2

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Embodiment Construction

[0034] Below in conjunction with accompanying drawing and embodiment the present invention will be further described:

[0035] 1 material

[0036] 1.1 Strains, plasmids, cells

[0037] Serum type 4 avian adenovirus JH was isolated and identified in our laboratory. DH5α competent cells, DH10Bac competent cells, and Sf9 insect cells, pFastBacHTA plasmids are all preserved by our laboratory

[0038] 1.2 Main reagents

[0039] The monoclonal antibody against F2 protein was kept by the laboratory; Phanta Super-Fidelity DNA polymerase, Gene1Kb Super Marker, 2×Taq Master Mix and ExnaseTMII ligase were purchased from Nanjing Novozyme Company; the plasmid mini-prep kit was purchased from AxyPrep Company ; Gel recovery DNA kit was purchased from QIAGEN; LipofectamineTM3000 transfection reagent and FBS fetal bovine serum were purchased from Gibco; goat anti-mouse FITC-labeled IgG was purchased from SIGMA; Western-blot exposure solution was purchased from Nanjing Feerte Biotechnology ...

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Abstract

The invention relates to a construction method of recombinant baculovirus for expressing serum type 4 avian adenovirus fibroid protein F2. The construction method comprises the following steps: (1) constructing an FAdV4F2 gene recombinant baculovirus expression vector; (2) packaging and passing baculovirus for expressing recombinant FAdV4 protein; and (3) transfecting baculovirus genome carrying FAdV4F2 gene into Sf9 cells, culturing in an incubator at 27 DEG C for 57 days, and observing cytopathic effect; when cytopathic effect reaches 70%, collecting cell lysis buffer, centrifuging to removeprecipitate, and preserving as seed virus; passing virus, and carrying out enlarged culture as seed virus when passing to the third generation. According to the invention, a baculovirus expression system is utilized to construct a recombinant baculovirus shuttle vector containing F2 gene, the recombinant baculovirus shuttle vector is transfected into Sf9 cells to obtain recombinant baculovirus rBAF2, and expression of recombinant protein F2 is identified by virtue of indirect immunofluorescence assay (IFA) and Western blot.

Description

technical field [0001] The invention relates to a method for constructing a recombinant baculovirus expressing the spike protein F2 of serotype 4 avian adenovirus, and belongs to the field of biotechnology. Background technique [0002] Fowl Adenovirus (Fowl Adenovirus, FAdV) belongs to Adenoviridae, and is divided into 5 species (A-E) and 12 serotypes. Among them, FAdV-4 can be transmitted horizontally and vertically through contact, and has the strongest pathogenicity to chicks, with a high lethality rate. The hosts of the disease include white-feathered broiler chickens, broiler breeders, laying hens, and yellow-feathered chickens. Clinical manifestations include inclusion body hepatitis (IBH) and hydropericardium hepatitis syndrome (HHS). With the rapid development of my country's chicken industry, the outbreak of avian adenovirus has caused serious economic losses to the poultry industry. The F2 protein is one of the capsid proteins of FAdV-4. The F2 protein has good ...

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Application Information

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IPC IPC(8): C12N15/866C12N15/70
CPCC12N15/86C12N15/70C12N2710/14043
Inventor 叶建强隆玉兰秦爱建张伟王伟康谢泉张建军李拓凡万志敏邵红霞
Owner YANGZHOU UNIV
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