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Method for detecting serum type 4 fowl adenovirus neutralizing antibody based on recombinant fluorescent virus

A technology of poultry adenovirus and detection method, which is applied in the field of biotechnology detection, can solve the problems of ineffective detection of FAdV-4 neutralizing antibody level, etc., and achieve the effects of clear fluorescent spots, reduced staining steps, and high biological safety

Pending Publication Date: 2022-01-11
YANGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But up to now, there is no report on using the recombinant FAdV-4 expressing the marker gene as the target virus of the neutralization test to realize the high-efficiency detection of the neutralizing antibody level of FAdV-4

Method used

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  • Method for detecting serum type 4 fowl adenovirus neutralizing antibody based on recombinant fluorescent virus
  • Method for detecting serum type 4 fowl adenovirus neutralizing antibody based on recombinant fluorescent virus
  • Method for detecting serum type 4 fowl adenovirus neutralizing antibody based on recombinant fluorescent virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Construction and identification of recombinant attenuated strain FAdV-4-EGFP expressing green fluorescent protein

[0035] CRISPR-Cas9 technology is used to construct the recombinant attenuated strain FAdV-4-EGFP expressing green fluorescent protein and its pathogenicity, in vivo and in vitro replication characteristics, see Chinese patent CN 112877303 A (paragraphs [0030]-[0044] of the description).

[0036] 2. Establishment of a new neutralizing antibody detection method based on FAdV-4-EGFP

[0037] a. Digest LMH cells in good condition on a 96-well plate and place at 37°C, 5% CO 2 Cultivate in a cell incubator, and prepare for testing when the cell density reaches about 80%;

[0038] b. Dilute the fluorescent virus to 20000TCID respectively 50 / 50μL, mixed with 1:8 pre-diluted negative and positive serum in 1.5mL EP tube at a volume ratio of 1:1, and incubated in a 37°C incubator for 1h;

[0039] c. Gently shake off the culture supernatant of the 96-well plate...

Embodiment 2

[0044] Other conditions are the same as in Example 1, the fluorescent virus infection amount is replaced by 1250, 2500, 5000 and 10000 TCID 50 / 50 μL / well, observe the growth of the virus under a fluorescent microscope, and take pictures for preservation. The result is as figure 1 shown. From figure 1 It can be seen that at 10000TCID 50 / 50μL / well and 20000TCID 50 At an infection volume of 50 μL / well, the number of EGFP-positive cells is more, which is more convenient for observation and avoids subjectivity.

Embodiment 3

[0046] Other conditions are the same as in Example 1, the culture time is changed to 12, 18, 30 and 36h, the growth of the virus is observed under a fluorescent microscope, photographed and recorded, the results are as follows figure 2 shown. From figure 2 It can be seen that at 24-36 hours, the number of EGFP-positive cells is relatively large, and a good detection effect has been achieved. Compared with the traditional neutralization test, the present invention can save 2-3 days and greatly improve the detection efficiency.

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Abstract

The invention discloses a method for detecting a serum type 4 fowl adenovirus neutralizing antibody based on a recombinant fluorescent virus. The method comprises the following steps: S1, diluting serum to be detected, mixing the diluted serum with 10000-20000 TCID50 / 50 [mu] L of fluorescent virus FAdV-4-EGFP, and carrying out constant temperature incubation for 1-2 h; s2, inoculating a mixture of the incubated virus and serum into LMH cells, culturing the mixture for 24-36 hours, and observing the neutralized condition of the fluorescent virus under a fluorescence microscope. On the basis of a recombinant virus FAdV-4-EGFP which is rescued in the early stage and expresses green fluorescent protein, the strain is a highly attenuated vaccine candidate strain, the chicken is not infected even if the chicken is infected by high-dose intramuscular injection, the growth characteristics of the strain are similar to those of a parent strain, the biological safety is high, and the risk of spreading of a strong virus does not exist. Compared with a traditional neutralization test, the scheme has the advantages that the result can be observed only by infecting the recombinant virus for 24 hours at least, the neutralization test result can be realized 2-3 days ahead of time, and the detection time is greatly shortened.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to a novel neutralizing antibody detection method based on a recombinant fluorescent virus strain FAdV-4-EGFP expressing green fluorescent protein. Background technique [0002] Fowl Adenovirus (Fowl Adenovirus, FAdV) belongs to the family Adenoviridae and is divided into 5 species (A-E) and 12 serotypes (1-7, 8a, 8b, 9-11). In recent years, the outbreak of hepatitis-pericardial effusion syndrome caused by highly pathogenic serotype 4 avian adenovirus (FAdV-4) has caused huge economic losses to the chicken industry at home and abroad. [0003] For the FAdV-4 epidemic, researchers have carried out vaccine research and development attempts, and the corresponding FAdV-4 inactivated candidate vaccine strains have been applied to poultry vaccination. The evaluation of the body's humoral immune response induced by the vaccine is an important indicator for evaluating the efficacy of...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/58G01N33/68
CPCG01N33/56983G01N33/582G01N33/6854G01N2333/075G01N2469/20
Inventor 郭艺文叶建强谢松桦张建军徐贞琦张伟谢泉万志敏李拓凡
Owner YANGZHOU UNIV
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