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Anti-peste des petits ruminants virus N protein monoclonal antibody and application thereof

A technology of Peste des petits ruminants and monoclonal antibodies, applied in the direction of antiviral immunoglobulin, application, biochemical equipment and methods, etc., can solve problems such as cumbersome operation, long detection cycle, and inability to meet the needs of rapid diagnosis of pathogens, and achieve Strong specificity and high biobinding activity

Inactive Publication Date: 2018-01-16
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are accurate and reliable, they cannot meet the actual work needs of rapid diagnosis of pathogens and timely control of the epidemic due to the disadvantages of cumbersome operation and long detection cycle. Therefore, the establishment of more sensitive and rapid detection technology has important practical significance for the prevention and treatment of this disease
In recent years, indirect immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies have been widely reported in the detection of this type of virus, but currently the sensitivity and specificity of Peste des petits ruminants virus are relatively strong The monoclonal antibody has not been reported

Method used

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  • Anti-peste des petits ruminants virus N protein monoclonal antibody and application thereof
  • Anti-peste des petits ruminants virus N protein monoclonal antibody and application thereof
  • Anti-peste des petits ruminants virus N protein monoclonal antibody and application thereof

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Embodiment 1

[0019] The preparation of embodiment 1 antigen

[0020] 1. PCR amplification of the N gene fragment of Peste des petits ruminants virus

[0021] According to the N gene sequence of Peste des petits ruminants virus (accession number GenBank: KM091959.1), primers were designed with the full length of the N gene of Peste des pets ruminants virus, and the cDNA of the N protein of Peste des pets ruminants virus was used as a template to amplify this virus by PCR. partial sequence. Wherein, in the amplified primer sequence, SalI and NotI are respectively added to the upstream and downstream primers.

[0022] Upstream primer (P1): 5'-GC GTC GAC TCATGGCGACTCTCCTTAAAAGC-3',

[0023] Downstream primer (P2): 5'-CGT GCGGCCGC GCCGAGGAGATCCTTGTCGTT-3',

[0024] The underlined part in the above primers is the restriction site.

[0025]

[0026] 2. Construction of a prokaryotic expression plasmid for the N protein gene fragment of Peste des petits ruminants virus

[0027] Peste de...

Embodiment 2

[0040] The preparation of embodiment 2 monoclonal antibody

[0041] 1. Immunity

[0042] The white mice used for immunization were 4-week-old female Balb / c mice (purchased from the Center for Disease Control and Prevention of Hubei Province). Purified recombinant proteins were used as antigens, and the immunization dose was 0.1 mg each time. Inject subcutaneously at multiple points with an interval of 14 days each time.

[0043]The first immunization: the purified recombinant protein was mixed with an equal amount of complete Freund's adjuvant (purchased from sigma company) as the antigen.

[0044] The second, third, and fourth immunizations: the purified recombinant protein was mixed with an equal amount of incomplete Freund's adjuvant (purchased from sigma) as the antigen.

[0045] Booster immunization three days before fusion: Purified recombinant protein was used as antigen and injected intraperitoneally.

[0046] 2. Cell Fusion

[0047] (1) Preparation of SP2 / 0 tumor ...

Embodiment 3

[0072] The biological activity detection of embodiment 3 monoclonal antibody

[0073] (1) Ascites titer determination of monoclonal antibody

[0074] specific method:

[0075] Use the indirect ELISA method to detect the ascites titer of the above-mentioned monoclonal antibody. After the PPRV-N-28a protein prepared by the present invention is used to coat the microtiter plate, the ascites is diluted from a volume ratio of 1:100 to 1:3276800 as a Anti-, HRP-labeled goat anti-mouse IgG (purchased from Boster Biological Co., Ltd.) was used as the secondary antibody for indirect ELISA detection, and a negative mouse serum control was set.

[0076] Results: The ascites titer of the monoclonal antibody of the present invention was 1:204800.

[0077] (2) Western blot detection

[0078] 1) When Vero cells were infected with PPRV for 96 hours, the cells showed obvious lesions. After the cells were lysed, 5×SDS was added (5×SDS preparation method: 1.25mL of 1M Tris-HCl, 0.5g of SDS, 2...

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Abstract

The invention belongs to the technical field of biological immunoassay, and more specifically relates to an anti-peste des petits ruminants virus N protein monoclonal antibody and an application thereof. The invention discloses the anti-peste des petits ruminants virus N protein monoclonal antibody, which is secreted by a hybridoma cell strain with a preservation number being CCTCC C2016135. The invention also discloses a preparation method and a purpose of the monoclonal antibody. The monoclonal antibody has strong specificity and good biological binding activity, can be reacted with Vero cells infected by peste des petits ruminants virus, and cannot be reacted with the normal Vero cells. The monoclonal antibody can be used for preparing a kit for detecting the peste des petits ruminantsvirus, is used for detecting peste des petits ruminants virus, and has the advantages of high detection specificity and high reaction sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biological immune analysis, and in particular relates to a monoclonal antibody of N protein of Peste des petits ruminants virus and an application thereof. Background technique [0002] Peste des petits ruminants (PPRV) mainly infects small ruminants such as goats, sheep, antelopes, and American white-tailed deer, and goats are more severely affected. The methods for detecting this virus mainly include agar gel immunodiffusion test (this method is simple, but the detection sensitivity of mild PPR with low viral antigen content is not high), immunocapture enzyme-linked immunosorbent assay (this method can quickly identify Diagnosis of Peste des petits ruminants virus and rinderpest virus), convective immunoelectrophoresis test, tissue culture and virus isolation (primary lamb kidney or African green monkey kidney cell tissue culture isolation can be used). Serological examination methods mainly include: v...

Claims

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Application Information

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IPC IPC(8): C12N15/45C07K14/115C07K16/10C12N5/20G01N33/577G01N33/569
Inventor 曹胜波郑云霞黄少梅刘学芹叶静魏燕鸣
Owner HUAZHONG AGRI UNIV
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