Infectious laryngotracheitis recombinant virus strain for expressing Newcastle disease virus F protein and establishment and application of strain

A technology of laryngotracheitis virus and Newcastle disease virus, which is applied in the direction of viruses, virus peptides, antiviral agents, etc., can solve the problems of exogenous immunogen gene construction that have not been reported, and achieve fast immune protection and good immune effect Effect

Active Publication Date: 2018-10-02
HARBIN WEIKE BIOTECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the construction and application of recombinant viruses using the US9 locus of the ILTV non-essential gene to carry out exogenous immunogen genes

Method used

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  • Infectious laryngotracheitis recombinant virus strain for expressing Newcastle disease virus F protein and establishment and application of strain
  • Infectious laryngotracheitis recombinant virus strain for expressing Newcastle disease virus F protein and establishment and application of strain
  • Infectious laryngotracheitis recombinant virus strain for expressing Newcastle disease virus F protein and establishment and application of strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Construction method of recombinant virus strain of infectious laryngotracheitis expressing Newcastle disease virus F protein.

[0049] (1) Construction of the recombinant transfer vector expressing EGFP protein:

[0050] Using the ILTV virus genome as a reference sequence for primer design, the GenBank accession number is NC_006623.1, and two pairs of primers were designed using Oligo 6.0 software to amplify the flanking sequences US9L and US9R of the US9 gene sequence. The primer sequences are as follows:

[0051] US9R-F: 5'-AAA CTGCAG ggctacacaacagcaata-3' (the underlined part is the restriction site of Pst I)

[0052] US9R-R: 5'-CGC GGATCC gaccaactaatagctatc-3' (the underlined part is the BamHI restriction site)

[0053] US9L-F: 5'-CGC GGATCCcagtaataataaccacac-3' (the underlined part is the restriction site of BamHI)

[0054] US9L-R: 5'-CGG GGTACC ctgtcctccgtccactct-3' (the underlined part is the Kpn I restriction site)

[0055] Use ILTV virus ...

Embodiment 2

[0122] Example 2. Protein expression and stability detection of recombinant virus expressing Newcastle disease F protein.

[0123] (1) The expression of Newcastle disease F protein was detected by indirect immunofluorescence (IFA).

[0124] IFA steps: 1. Inoculate LMH cells grown into a monolayer on a six-well plate with a low dose of recombinant virus, and wash three times with PBS 24 hours after inoculation. At the same time, set parental virus and LMH cell blank control.

[0125] 2. Fix with 4% polymethanol, about 1ml per well, and act for 30min at room temperature. Wash three times with PBS.

[0126] 3. Add 0.5ml / well of 1:100 times diluted positive serum (diluted with PBS) to the primary antibody, and act at 37°C for 1h. Wash three times with PBS.

[0127] 4. Add 1:320 dilution of rabbit anti-chicken IgY FITC antibody (Sigma, 0.2ml / well) to the secondary antibody, and act at 37°C for 1h. Wash three times with PBS.

[0128] 5. Observe the results under a fluorescence...

Embodiment 3

[0136] Example 3. Application evaluation of the recombinant virus of infectious laryngotracheitis stably expressing Newcastle disease F gene.

[0137] 1. Test material

[0138] 1.1 The recombinant virus of the rILTV△US9-NDV-F strain was constructed by our research team and propagated in LMH cells. The virulent Newcastle disease Beijing strain and the virulent infectious laryngotracheitis strain Wanggang strain were preserved by our research team.

[0139] 1.2 SPF test chicken: from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0140] 2. Test method

[0141] Inoculate 20 28-day-old SPF chickens with 0.1ml of rILTV△US9-NDV-F recombinant virus, and the other 20 as controls. 21 days after immunization, 10 immunized chickens and 10 control chickens were challenged with strong Newcastle disease. Poison Beijing strain. At the same time, another 10 immunized chickens and 10 control chickens were challenged with W...

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Abstract

The invention discloses an infectious laryngotracheitis recombinant virus strain for expressing a Newcastle disease virus F protein and establishment and the application of the strain and relates to the field of recombinant virus vaccines. Aiming at problems of recombinant virus establishment and the application of an exogenous immunogenic gene with a nonobligatory gene US9 locus of an ILTV (Infectious Laryngotracheitis Virus), the invention provides an infectious laryngotracheitis recombinant virus strain which is named as rILTV US9-NDV-F, the microorganism preservation number of the strain is CGMCC No.14738, and the virus strain is an infectious laryngotracheitis recombinant virus which is established by replacing a US9 gene of the infectious laryngotracheitis recombinant virus by the Newcastle disease virus F protein, establishing an acquirement deficiency US9 gene and by inserting an NDV-F (Newcastle Disease Virus-F Protein) expression box into a corresponding position of the gene,and is used for recombining the Newcastle disease virus F protein. The recombinant infectious laryngotracheitis recombinant virus strain disclosed by the invention is used for preparing vaccines forpreventing infectious laryngotracheitis and other chicken infectious diseases.

Description

technical field [0001] The invention relates to the field of recombinant virus vaccines, in particular to an infectious laryngotracheitis recombinant virus strain expressing Newcastle disease virus F protein and its construction method and application. Background technique [0002] Infectious laryngotracheitis (ILT) is an acute viral infectious disease of the upper respiratory tract of chickens caused by infectious laryngotracheitis virus (ILTV). Infectious laryngotracheitis virus mainly infects the chicken respiratory system, especially the tracheal epithelium is the main breeding place of the virus. Infected chickens show symptoms such as mouth breathing, hemoptysis, conjunctivitis, and tears during the onset of the disease, and the fatality rate of some virulent infections reaches 100%. After the disease was first reported in the United States in 1925, it has now spread to many chicken farming areas in the world. The disease spreads quickly and has a high mortality rate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K39/295A61K39/245A61K39/17A61P31/22A61P31/14
CPCA61K39/12A61K2039/552A61K2039/70A61P31/14A61P31/22C07K14/005C12N7/00C12N2710/16021C12N2710/16034C12N2760/18122C12N2760/18134
Inventor 刘胜旺邵昱昊孙军峰韩宗玺
Owner HARBIN WEIKE BIOTECH DEV
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