Serum type 1 Marek's disease virus MEQ monoclonal antibody, and preparation method and application thereof

A monoclonal antibody and Marek's disease technology, applied in the field of animal immunology, can solve the problems of large protein, limited research needs of scholars, failure to develop MEQ antibody, etc., and achieve accurate and strong specificity

Active Publication Date: 2022-05-13
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]Although domestic scholars have tried many times to develop antibodies to this protein, due to factors such as the high GC content of the meq gene sequence, the large protein size, and the difficulty in artificial expression and purification , my country has not yet been able to develop a practical MEQ antibody, and there are no commercial products available on the market
At present, only the World Organization for Animal Health (OIE) MDV Reference Laboratory of the United States Department of Agriculture's Poultry Disease and Tumor Laboratory and the British Pirbright Institute have successfully developed a monoclonal antibody for this protein, but no commercial use has been made. Chinese scholars carry out relevant research, and the antibodies needed can only rely on the friendship gifts of European and American scholars from these two institutions, which greatly limits the research needs of Chinese scholars

Method used

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  • Serum type 1 Marek's disease virus MEQ monoclonal antibody, and preparation method and application thereof
  • Serum type 1 Marek's disease virus MEQ monoclonal antibody, and preparation method and application thereof
  • Serum type 1 Marek's disease virus MEQ monoclonal antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Design and immunogenicity identification of the hydrophilic polypeptide of the MEQ protein of serotype I Marek's disease virus

[0047] 1. Comparative analysis of amino acid difference sites and hydrophilicity of MEQ protein of different pathogenic MDV-1 strains

[0048] The meq gene sequences of different pathogenic strains of MDV-1 (including the virulent strain GA, the super-virulent strain GX0101, Md5 and the super-virulent strain 648A) were downloaded from the NCBI GenBank database. Using DNAStar MegAlign software to carry out gene sequence comparison analysis, it can be seen that the MEQ protein sequences of the four different pathogenic MDV strains are very highly conserved, and there are only 11 amino acid site differences between them ( figure 1 and Table 1). The above analysis results provide an important basis for the design and preparation of broad-spectrum MEQ protein monoclonal antibodies that can recognize different pathogenic MDV-1 strains. ...

Embodiment 2

[0064] Example two, screening and identification of anti-MDV-1MEQ protein monoclonal antibody

[0065] 1. Cell fusion and establishment of hybridoma cell lines

[0066] The No. 2 mouse with the highest IFA titer was selected for cell fusion. Before fusion, intraperitoneally inject 50 μg of 4 kinds of polypeptide-KLH coupling proteins mixed in equal ratio without adjuvant for superimmunization, and after 3 days of superimmunization, mouse splenocytes are taken for cell fusion, and the SP20 cells are combined with superimmunization The splenocytes of immune mice were counted and mixed according to the ratio of 1:10, and fused with PEG-1500 fusion agent. The fused cells were resuspended in RPMI-1640 / HAT medium and seeded in four 96-well cell culture plates. medium, 200 μL / well, placed at 37°C, 5% CO 2 After culturing in an incubator, after about 7-10 days, a large hybridoma cell stack was grown, and the cell supernatant was collected for the following IFA detection.

[0067] 2...

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Abstract

The invention discloses an anti-serum type 1 Marek's disease virus MEQ protein monoclonal antibody as well as a preparation method and application thereof. The anti-MDV-1 specific MEQ protein monoclonal antibody is generated by a mouse monoclonal antibody hybridoma cell strain meq-2A9-B12, the cell strain is preserved in the China General Microbiological Culture Collection Center, the preservation number is CGMCC No: 23023, the subtype of the monoclonal antibody is IgG2b, the light chain type of the monoclonal antibody is Kappa, the amino acid sequence of a heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.3, and the amino acid sequence of a heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.4. The monoclonal antibody can be used for detecting the MDV-1 specific MEQ protein. And the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.4. The monoclonal antibody 2A9-B12 secreted by the invention can be used for immunological detection analysis of IFA, Western blot and the like of strains with different pathotypes of MDV-1, can also be used for identifying and distinguishing MDV-2 and MDV-3 strains, and is suitable for differential diagnosis of MDV epidemic strains with different serotypes in clinical suspected cases. The invention solves the problem of'neck 'of lack of anti-MDV-1 MEQ protein specific monoclonal antibody in China at present, and provides a key core reagent for subsequent research of MD and research and development of diagnostic reagents.

Description

technical field [0001] The invention relates to a monoclonal antibody against serotype 1 Marek's disease virus MEQ, a preparation method and application thereof, and belongs to the technical field of animal immunology. Background technique [0002] Chicken Marek's disease (Marek's disease, MD) is an important immunosuppressive disease and tumor disease caused by early infection of chickens by Marek's disease virus (MDV). Severe immunosuppression after MDV infects the host in the early stage is one of the important factors for the poor immune prevention effect of clinical poultry disease vaccines. It has long plagued the healthy development of my country's poultry industry. At the same time, MDV will induce visceral tumors and a large number of deaths after infecting natural hosts, resulting in an average annual direct economic loss of about US$1-2 billion in the global poultry industry. The occurrence of MDV-induced host tumors can be effectively prevented by vaccines. It i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/08G01N33/569G01N33/574G01N33/577G01N33/68C12N15/85C12N15/89
CPCC07K16/085G01N33/577G01N33/56994G01N33/57484G01N33/68C12N15/89C12N15/85C07K2317/20C07K2317/56G01N2333/03Y02A50/30
Inventor 罗俊滕蔓刘金玲张改平罗琴郑鹿平邢广旭卢清侠赵东柴书军
Owner HENAN ACAD OF AGRI SCI
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