Infectious bronchitis recombinant virus lack of E protein ion channel activity as well as preparation method and application

A bronchitis, ion channel technology, applied in the field of molecular biology, can solve problems such as titer decrease, apoptosis increase, virion morphology defects, etc.

Active Publication Date: 2019-03-26
SOUTH CHINA AGRI UNIV
View PDF3 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some coronaviruses, the recombinant virus that knocks out the E gene can still maintain the ability to replicate, but its virion form has serious defects, and the titer is significantly reduced. For example, in SARS-CoV, the deletion of the E protein reduces the Pathogenicity and mortality, while recombinant SARS-CoV lacking the E gene led to increased apoptosis and down-regulation of inflammatory factors associated with infected cells

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Infectious bronchitis recombinant virus lack of E protein ion channel activity as well as preparation method and application
  • Infectious bronchitis recombinant virus lack of E protein ion channel activity as well as preparation method and application
  • Infectious bronchitis recombinant virus lack of E protein ion channel activity as well as preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1 IBV infectious cDNA cloning based on seamless cloning technology

[0097] Based on seamless technology, the mutation design of the E protein gene locus sequence of infectious bronchitis virus was carried out, and 5 fragments across the entire IBV genome were obtained, namely fragment A, fragment B, fragment C, fragment D and fragment E. Restriction sites were introduced into the 5'end and 3'end of the 5 fragments, combined with the T7 promoter, and ligated in vitro to obtain a cloned strain, and transcribed in vitro to obtain a full-length RNA (such as figure 1 Shown); The specific steps are as follows:

[0098] (1) Use chicken IBV to infect African green monkey kidney cells (Vero cells, ATCC). For the source and infection method of IBV, please refer to 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.".

[0099] (2) The...

Embodiment 2

[0114] Example 2 The recombination and purification method of rIBV-A26F lacking the activity of the E protein ion channel includes the following steps:

[0115] (1) First, use seamless cloning technology to design the deletion of the E protein gene site sequence of infectious bronchitis virus, combine with the T7 promoter, and connect in vitro to obtain a complete RNA clone; then the above clone is cloned for infectious, The cloned sequence to be obtained is introduced into Vero cells by electrotransformation method to obtain recombinant infectious bronchitis virus lacking the activity of the E protein ion channel: the specific operation is the same

[0116] Example 1.

[0117] (2) Collect the cell supernatant infected with rIBV-A26F, and then dilute it 10 times with serum-free DMEM, and test the virus titer by plaque purification experiment.

[0118] (3) Infect the diluted supernatant virus into a plate paved with Vero cells in a 6-well plate. After 2 hours, the virus is adsorbed, th...

Embodiment 3

[0124] Example 3 Stability analysis of attenuated strains of IBV mutants adapted to the lack of E protein ion channel activity of Vero cells

[0125] (1) Infect Vero cells with rIBV-A26F mutant virus, culture at 37°C for 24 hours, collect the supernatant, and then use the supernatant virus to infect newly plated Vero cells, and so on to infect 30 generations;

[0126] (2) Collect, lyse and extract the cellular RNA infected with the virus at generations 5, 10, 15, 20, 25, and 30, RT-PCR, E gene sequencing, and sequence comparison to see if there are any new mutations in the E gene. To determine the genetic stability of the strain, the primers of the amplified E gene are the same as those in Table 2, and the sequencing primers are the upstream primers.

[0127] (3) Analysis of sequencing results. Starting from the 10th generation, the second T of the 26th amino acid phenylalanine of the rIBV-A26F mutant virus was gradually replaced with C, and the original TTT codon was converted to TC...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses an infectious bronchitis recombinant virus lack of E protein ion channel activity as well as a preparation method and application. A 26-site amino acid of a protein E of a wildinfectious bronchitis recombinant virus is mutated from aminopropionic acid to phenylalanine to obtain the infectious bronchitis recombinant virus lack of the E protein ion channel activity. By constructing the infectious bronchitis recombinant virus lack of the E protein ion channel activity and obtaining a mutant as well as by virtue of a separation purification method for the mutated virus andthe characteristic analysis of the mutated virus, the development of specific antiviral drugs and effective vaccines for preventing the virus infection can be facilitated, and the important public health and economic significance for researching corona virus can be achieved.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and particularly relates to an infectious bronchitis recombinant virus lacking E protein ion channel activity, and a preparation method and application. Background technique [0002] Chicken infectious bronchitis virus is a coronavirus, which was first isolated in 1937. This coronavirus caused significant economic losses to the poultry industry worldwide. Coronavirus is highly contagious, causing diseases of the respiratory tract, kidneys and fallopian tubes, causing the body to become lighter, the kidneys malfunction, and the number and quality of eggs laid. Related studies have shown that coronaviruses can cross the species barrier and become the pathogen of deadly zoonotic diseases. Currently, there is no effective treatment for coronavirus infection, and the development of attenuated vaccines and specific antiviral drugs is a promising strategy. [0003] The E protein is the key to the pathogenici...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/50C12N15/63
CPCC12N7/00C12N15/63C07K14/005C12N2770/20021C12N2770/20022
Inventor 冯涛声刘定祥陈瑞爱李淑敏李延鹏梁佳琪袁丽霞朱庆春罗琼熊挺
Owner SOUTH CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap