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Avian metapneumovirus (aMPV) F protein polypeptide and application thereof

A technology of protein polypeptide and carrier protein, which is applied in the field of avian metapneumovirus F protein polypeptide and detection of avian metapneumovirus infection, can solve the problems that have not been reported, and achieve the effect of high sensitivity, stable sensitivity and high sensitivity

Active Publication Date: 2015-07-22
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the monoclonal antibody against avian metapneumovirus has been reported to be a monoclonal antibody prepared by subgroup C virus. A total of 6 strains have been obtained, all of which are prepared against the virus N protein, and no monoclonal antibody to other proteins has been reported.

Method used

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  • Avian metapneumovirus (aMPV) F protein polypeptide and application thereof
  • Avian metapneumovirus (aMPV) F protein polypeptide and application thereof
  • Avian metapneumovirus (aMPV) F protein polypeptide and application thereof

Examples

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Embodiment 1

[0028] The determination of embodiment 1 avian metapneumovirus F protein antigen short peptide

[0029] According to the protein sequence of the F gene of avian metapneumovirus, bioinformatics analysis and comparison were carried out to determine the following polypeptide: CTNAGSTAYYPNKDD was named as the F protein polypeptide as the short antigenic peptide. The antigen short peptide has high conservation and strong antigenicity, and was synthesized by Shanghai Jier Biochemical Company. The coupling is based on the maleimide theory, that is, the cysteine ​​residue in the polypeptide sequence and the amino group in the carrier protein coupling. Therefore, when designing a polypeptide antigen, a cysteine ​​residue is often added to its N-terminal or C-terminal to facilitate the coupling of the polypeptide to the carrier protein.

Embodiment 2

[0030] The preparation of embodiment 2 monoclonal antibody

[0031] 1) Peptide conjugation and animal immunization

[0032] Peptides were conjugated to KLH (Pierce Cat#77611) and BSA (AMRESCO code0332). After desalting, the KLH-coupled peptide was mixed with an equal amount of CFA or IFA as an immunogen and stored at 4 degrees. Three BALB / c mice were immunized with the immunogen mixed with CFA on day 0, and then boosted with the immunogen mixed with IFA several times in the following days. On the 14th day, the tail blood of the mice was drawn, coated with uncoupled polypeptide and carrier protein BSA-coupled polypeptide, and the corresponding titers were detected by ELISA to observe the immune response against the antigen.

[0033] The detection method of ELISA is: add 100ng detection original to each well of 96-well plate (Corning, USA), wrap the plate overnight at 4°C. After washing twice with phosphate buffered saline (PBS), block with 5% skimmed milk powder PBS for one ...

Embodiment 3

[0060] Example 3 The IFA method and immunoblotting (WB) method of monoclonal antibody preparation are used for the detection of avian metapneumovirus infection

[0061] The monoclonal antibody produced by the monoclonal antibody strain 2B8 was used to detect the infection of avian metapneumovirus on Vero cells, using indirect immunofluorescence (IFA) and immunoblotting (WB) methods for detection. The IFA detection method is as follows: Vero cells grown into a single layer in a 96-well cell plate were washed twice with phosphate buffered saline (PBS), then inoculated with avian metapneumovirus, and cultured at 37°C in an incubator containing 5% CO2; At a given time point after virus inoculation, fix with 4% paraformaldehyde at room temperature for 30 minutes; after washing, the cell wells were washed with 1:30 times diluted anti-Avian metapneumovirus F protein monoclonal antibody (the dilution solution is 3%BSA- PBS) at 37°C for 90 minutes; after washing, the cell wells were in...

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Abstract

The invention provides an F protein polypeptide and an application thereof. The antigen polypeptide is obtained through bioinformatics analysis and comparison according to F protein gene sequences. The amino acid sequence of the antigen polypeptide is CTNAGSTAYYPNKDD. The antigen polypeptide has high conservative property and strong antigenicity, and monoclonal antibodies aiming at the Avian metapneumovirus (aMPV) F proteins can be generated by utilizing the polypeptide.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an avian metapneumovirus F protein polypeptide, and also relates to the use of the protein polypeptide in detecting avian metapneumovirus infection. Background technique [0002] Avian metapneumovirus (aMPV) infection is a new infectious disease that endangers the healthy development of the poultry industry. The pathogen aMPV belongs to the Pneumovirus genus of the Paramyxoviridae Pneumoviridae subfamily and has no hemagglutination and neuraminidase properties. aMPV can widely cause disease in chickens, turkeys, pheasants, and guinea fowls, and antibodies can also be detected in wild birds. Sick poultry infected with aMPV often have mild or severe respiratory symptoms, decreased feed intake and egg production. Many chickens have swollen heads, and aMPV is the main pathogen that induces swollen head syndrome (SHS); while turkeys mainly present with acute respiratory symptoms—turkey r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K19/00C07K16/10G01N33/577G01N33/569
Inventor 王菁刘爵韦莉朱珊珊张春燕阎旭全荣李子璇
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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