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Infectious bronchitis virus with mutation of glycosylation site of M protein and preparation method and application thereof

A technique for bronchitis and site mutation, applied in biochemical equipment and methods, chemical equipment and methods, botany equipment and methods, etc.

Inactive Publication Date: 2019-03-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, emerging IBV variants have emerged, but an effective IBV vaccine that provides broad protection has not yet been successfully developed. Therefore, the study and understanding of IBV as an important pathogen is critical

Method used

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  • Infectious bronchitis virus with mutation of glycosylation site of M protein and preparation method and application thereof
  • Infectious bronchitis virus with mutation of glycosylation site of M protein and preparation method and application thereof
  • Infectious bronchitis virus with mutation of glycosylation site of M protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1IBV

[0053] Construction of embodiment 1 IBV M protein mutant strain N3D / N6D full-length infectious clone

[0054] Construct 5 plasmids containing 5 fragments (A to E) spanning the entire IBV genome, such as figure 1 , and BsmBI or BsaI restriction sites were introduced into the 5' and 3' ends of the fragment. In fragment A, the T7 promoter sequence is inserted upstream of the 5' end of the IBV genome, and T7 polymerase is used to promote in vitro transcription. The specific steps are as follows:

[0055] (1) Infect African green monkey kidney cells (Vero cells, ATCC) with chicken IBV. For the source and infection method of IBV, see the literature "S. Shen, et al. Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein inpathogenesis and replication.”.

[0056] (2) The total RNA extracted from IBV-infected African green monkey kidney cells (Vero cells, ATCC) by TrizoL was amplified by RT-PCR technique, ...

Embodiment 2

[0067] Embodiment 2Western blot verifies the expression of mutant virus strain protein

[0068] The Vero cells infected with the mutant IBV prepared in Example 1 were lysed with 1× RIPA buffer solution (Beiyuntian) to obtain the total protein, which was mixed with a 5-fold concentration of loading dye containing 0.1M DTT Afterwards, the cell lysate was boiled at 95°C for 5 minutes and processed by SDS-PAGE. protein in PBST buffer containing 5% skimmed milk powder (80mM Na 2 HPO 4 , 20mM NaH 2 PO 4 , 100mM NaCl, 0.1% Tween 20, pH7.5), incubated overnight at 4°C; the membrane was incubated with IBV-M or IBV-N antibody (M protein or N protein was expressed in BL21 competent (Tiangen) prokaryotic Purified and injected into rabbits, M antibody and N antibody have been disclosed in the literature "Sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus") incubated for 1 hour, washed with PBST 3 times, and horseradish peroxidase (Beijing Quanquan ...

Embodiment 3

[0070] Embodiment 3 measures virus proliferation curve

[0071] Divide Vero cells by 1 × 10 5 cells / mL were inoculated on a 6-well plate (1mL cell suspension was inoculated in each well), and Vero cells were infected with wild-type IBV and mutant IBV at an MOI of 1, and harvested at different times after infection, and the virus was obtained after freezing and thawing three times The stock solution was then serially diluted 10 times to infect Vero cells on a 96-well plate. On the 3rd day after infection, the number of infected wells was read according to cytopathy (cell fusion), and the half cell infection amount (TCID) of each sample was determined by the Reed and Muench method. 50 ).

[0072] The result is as Figure 4 As shown, the virus proliferation curve shows that there is no significant difference in the proliferation of the M protein mutant and wild-type IBV strains on the cells.

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Abstract

The invention discloses an infectious bronchitis virus with the mutation of a glycosylation site of a M protein, a preparation method and application thereof. According to the preparation method, a full-length infectious clone of the IBV (infectious bronchitis virus) is obtained by utilizing a reverse genetics technology; then the N->D point mutation is carried out on the glycosylation site of theM protein of the IBV by using the full-length infectious clone as a template; and the mutated virus stain of the M protein of the IBV is obtained after the in vitro transcription of electrotransfection cells. The obtaining of the mutant strain is conductive to studying the influence of the mutation of the glycosylation site of the M protein of the IBV on the structure of the M protein, the proliferation of the virus and the endoplasmic reticulum pressure, to judging whether the replication capacity and pathogenicity of the virus change and to providing a reference method for the development of IBV attenuated vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an infectious bronchitis virus with a mutated M protein glycosylation site, a preparation method and application thereof. Background technique [0002] Infectious bronchitis virus (IBV) can cause acute and highly contagious infectious diseases in chickens, and is one of the main pathogens affecting the global poultry industry. Since it was first isolated in 1931, a surprising number of IBV variants have been found around the world body. In recent years, emerging IBV variants have emerged, but an effective IBV vaccine that can provide broad protection has not been successfully developed. Therefore, the study and understanding of IBV as an important pathogen is critical. In fact, with IBV as the prototype of coronaviruses, research in the past few decades has revealed some of the most basic concepts in molecular cell biology and the pathogenesis of coronaviruses, but there...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/50C12N15/63A61K39/215A61P31/14
CPCA61K39/12A61K2039/5254A61K2039/552A61P31/14C07K14/005C12N7/00C12N15/63C12N2770/20021C12N2770/20022C12N2770/20034
Inventor 刘定祥冯涛声陈瑞爱梁佳琪李延鹏李淑敏袁丽霞朱庆春罗琼熊挺
Owner SOUTH CHINA AGRI UNIV
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