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Construction method of recombinant baculovirus for expressing novel goose astrovirus capsid protein ORF2

A technology of recombinant baculovirus and astrovirus, applied in the direction of virus, viral peptide, virus/bacteriophage, etc., to achieve the effect of high protein expression, good immunogenicity and reactogenicity, and easy purification

Pending Publication Date: 2022-05-10
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no serological method for rapid and specific detection of GAstV to commercialize GAstV vaccines. In order to reduce the losses caused by GAstV to the goose industry in my country, it is necessary to further study the establishment of diagnostic methods and the production of GAstV-ORF2 subunit vaccines.

Method used

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  • Construction method of recombinant baculovirus for expressing novel goose astrovirus capsid protein ORF2
  • Construction method of recombinant baculovirus for expressing novel goose astrovirus capsid protein ORF2
  • Construction method of recombinant baculovirus for expressing novel goose astrovirus capsid protein ORF2

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Embodiment Construction

[0038] The present invention will be further described below in conjunction with the embodiments and accompanying drawings.

[0039] 1 material

[0040] 1.1 Strains, plasmids, cells

[0041] The GAstV-GD strain was isolated and identified by our laboratory. DH5α competent cells, DH10Bac competent cells, and Sf9 insect cells were all preserved in our laboratory, and the pFastBacHTA plasmid was purchased from Invitrogen.

[0042] 1.2 Main reagents

[0043]The monoclonal antibody against ORF2 protein was prepared and stored in the laboratory; RNA extraction kit, RNA reverse transcription reagent, Phanta Super-Fidelity DNA polymerase, Gene 1Kb Super Marker, 2×Taq Master Mix and ExnaseTMII ligase were purchased from Nanjing Nuo Weizan Company; plasmid mini-extraction kit was purchased from AxyPrep Company; gel recovery DNA kit was purchased from QIAGEN Company; LipofectamineTM 3000 transfection reagent and FBS fetal bovine serum were purchased from Gibco Company; goat anti-mouse...

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Abstract

The invention relates to a construction method of a recombinant baculovirus for expressing a novel goose astrovirus capsid protein ORF2, which comprises the following steps: (1) constructing a GAstV-ORF2 gene recombinant baculovirus expression vector; (2) packaging and passaging the baculovirus for expressing the recombinant GAstV-ORF2 protein; (3) transfecting a baculovirus plasmid carrying the GAstV-ORF2 gene into an Sf9 cell, culturing in a CO2-free incubator at 27 DEG C for 5-7 days, and observing the cytopathy condition every day; when the cytopathic effect reaches 70%, collecting cell supernatant, and storing at-80 DEG C; and when the third generation is passed, storing the virus as a seed virus, and carrying out enlarged culture to obtain the recombinant baculovirus for expressing the novel goose astrovirus capsid protein ORF2. According to the principle, the baculovirus capable of expressing the capsid protein ORF2 is obtained by amplifying a full-length fragment of the goose astrovirus capsid protein ORF2, constructing a baculovirus recombinant vector and transfecting an Sf9 cell.

Description

technical field [0001] The invention relates to a method for constructing a recombinant baculovirus expressing novel goose Astrovirus capsid protein ORF2, belonging to the field of biotechnology. Background technique [0002] Goose Astrovirus (GAstV) belongs to the family Astroviridae and the genus Avian Astrovirus. GAstV can cause goose gout symptoms with a large amount of urate deposition on the surface of internal organs such as the liver, spleen and kidneys, and in the joint cavity. It is highly pathogenic to goslings and has a high lethality rate. With the rapid development of the goose industry in my country, the outbreak of goose astrovirus has caused serious economic losses to the goose industry. However, there is currently no serological method for rapid and specific detection of GAstV to commercialize GAstV vaccines. In order to reduce the losses caused by GAstV to the goose industry in my country, it is necessary to further study the establishment of diagnostic m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/40C12N7/01
CPCC12N15/86C07K14/005C12N7/00C12N2770/12022C12N2710/14021C12N2710/14043C12N2800/105
Inventor 高巍张辛耘叶建强任丹秦爱建万志敏李拓凡谢泉邵红霞
Owner YANGZHOU UNIV