Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof

A technology of African swine fever virus and monoclonal antibody, which is applied in a field, can solve the problems of chromosome loss, obstruction, and lack of secretion of single antibody, etc., and achieve good immunogenicity, shorten test time, and simple operation steps.

Active Publication Date: 2021-08-03
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Secondly, after immunization with high-quality antigens, there are obstacles in the process of cell fusion and positive clone cell selection. For example, although the success rate of cell fusion is high, the successfully fused cells do not have the ability to secrete a single antibody; During the process, the status of some hybridoma cells was unstable. After multiple subcloning, the chromosomes were lost and could not continue to secrete a single antibody; after the hybridoma cells secreting a single antibody were obtained, ascites was obtained by immunizing mice, and the ascites titer obtained Not high; and after some monoclonal antibodies are prepared, because the recognized target is inconsistent with the serum to be tested, it is impossible to establish a blocking Elisa detection method

Method used

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  • Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof
  • Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof
  • Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Preparation of anti-ASFV p54 monoclonal antibody and obtaining secreted hybridoma cell lines:

[0023] The antigen of the present invention is African swine fever virus truncated protein p54 (shown in SEQ ID NO.1), and the indirect ELISA detection kit prepared by using the protein as antigen coating can accurately detect whether the sample contains African swine fever virus At the same time, compared with the Spanish detection kit recommended by OIE, the specificity of the indirect ELISA kit established by this antigen is comparable to it, and the sensitivity is better than that of the Spanish detection kit.

[0024] 1. Animal immunity

[0025] Get 200 μ g of African swine fever virus truncated protein p54 (shown in SEQ ID NO.1) and Freund's complete adjuvant of the same volume, use a syringe after emulsifying with an emulsifier to carry out multiple injections on the back of 6-8 week old BALB / c mice Point injection, 14 days later for the second immunization. Second i...

Embodiment 2

[0035] Example 2: Preparation of ascites and identification of monoclonal antibody characteristics

[0036] 1. Ascites preparation

[0037] Take 8-week-old BALB / c mice, intraperitoneally inject Freund's incomplete adjuvant, 0.5mL / mouse; 7-10 days later, intraperitoneally inject hybridoma cells 4-4C in logarithmic growth phase diluted with basal medium, 1.2×10 6 Individual / only; observe the state of the mice. After 7-14 days, the abdomen of the mouse was obviously bulged, and the ascites was collected from the abdominal cavity with a 16# needle, centrifuged at 1500r / min for 10min, the fat tissue was removed, the supernatant was absorbed, and stored at -20°C for later use.

[0038] 2. Characterization of monoclonal antibody

[0039] 2.1 Potency determination

[0040] The recombinant protein p54 was coated, placed overnight at 4°C, washed 3 times with PBST, and blocked with blocking solution. The coated plate was placed in a 37°C incubator, incubated for 2 hours, washed 3 tim...

Embodiment 3

[0048] Example 3: Identification of reactivity of monoclonal antibodies with whole virus antigens

[0049] 3.1 ELISA method to detect the reactogenicity of ASFV and obtained ascites

[0050] Dilute the purified African swine fever whole virus (purified virus concentration is 0.8mg / mL) at 1:800, coat at 4°C overnight, wash 3 times with PBST, seal the coated plate with blocking solution, and place Incubate at 37°C for 2 hours. The ascites obtained in Example 2 was diluted 2 times starting from 1:100 until 1:12800, and negative and positive controls were set at the same time, 100 μl per well, placed in a 37°C incubator, incubated for 30min, washed 5 times with PBST, and added to the working Goat anti-mouse HRP-IgG with a concentration of 1:5000, placed in a 37°C incubator, incubated for 30 minutes, washed 5 times with PBST, added TMB to avoid light for 10 minutes, hydrofluoric acid terminated the reaction, and read OD 630nm . The conditions for the establishment of this method...

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Abstract

The invention belongs to the technical field of clinical immunology and discloses a monoclonal antibody prepared from the truncated protein p54 of African swine fever virus and its application. The present invention finally obtains the monoclonal antibody 4-4C by immunizing the effectively truncated African swine fever virus protein p54 and systematically screening the hybridoma technology. The obtained monoclonal antibody can react with the whole virus of African swine fever and meet the basic requirements of monoclonal antibodies; in addition, the specificity of the monoclonal antibody is good, and there is no cross-reaction with other proteins of African swine fever virus; moreover, the monoclonal antibody After the cloned antibody passed the Elisa test, its titer was as high as 1,600,000; finally, the monoclonal antibody had a blocking effect, and the established blocking Elisa detection method had the same detection effect as the Spanish detection kit recommended by OIE, and shortened the detection time , has the ability to detect the level of African swine fever antibody for pig farms.

Description

technical field [0001] The invention belongs to the technical field of clinical immunology, relates to the technical field of detection and identification of African swine fever virus, in particular to a monoclonal antibody prepared from the truncated protein p54 of African swine fever virus, and also relates to a monoclonal antibody secreting African swine fever virus The hybridoma cell of cloning antibody also relates to the application of the monoclonal antibody prepared by truncating protein p54 in the preparation and detection of African swine fever virus kit. Background technique [0002] African swine fever is an acute and highly contagious zoonotic disease of pigs caused by African swine fever virus. It has the characteristics of high incidence, short course of disease and high mortality. African swine fever virus belongs to the African swine fever virus family, double-stranded DNA virus, its genome is large, 170-190bp, and the gene is variable, there is a variable r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/08G01N33/569G01N33/577
CPCC07K16/081C07K2317/35G01N33/56983G01N33/577G01N2333/01
Inventor 金梅林杨永张强赵亚孙小美吕长杰杨丽
Owner HUAZHONG AGRI UNIV
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