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Indirect ELISA kit for detecting feline coronavirus antibody

A feline coronavirus and kit technology, applied in viral peptides, biological testing, introduction of foreign genetic material using vectors, etc., can solve the problems of high S1 functional domain variability and unfavorable target protein regions, and achieve strong specificity and sensitivity. High and stable effect

Active Publication Date: 2021-08-03
LONGYAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the S1 functional domain contains the receptor binding region RBD and a large number of epitope regions, but the S1 functional domain has high variability, which is not conducive to being used as a target protein region; while the S2 functional domain is a relatively conserved part of the S protein, which mediates virus Fusion with cell membrane, and rich in epitopes

Method used

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  • Indirect ELISA kit for detecting feline coronavirus antibody
  • Indirect ELISA kit for detecting feline coronavirus antibody
  • Indirect ELISA kit for detecting feline coronavirus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cloning sequencing analysis, expression and purification of embodiment 1 feline coronavirus S recombinant protein

[0033] (1) Primer design and synthesis

[0034] Referring to the FCoV S protein gene sequence published in GenBank, a pair of specific primers were designed and synthesized: the upstream primer sequence is shown in SEQ ID NO:2; the downstream primer sequence is shown in SEQ ID NO:3. The double enzyme cutting sites in the downstream primers were designed as NdeI and XhoI respectively. Primers were synthesized, purified and sequenced by Shanghai Sangon Bioengineering Co., Ltd.

[0035] (2) PCR using the above primers

[0036] Feline coronavirus S protein gene was used as template, the amplification system was 25 μL, including 12.5 μL of 2×TransStart FastPfuPCR SuperMix, 1 μL of upstream and downstream primers, 1 μL of plasmid template, supplemented with sterilized ddH 2 O to a final volume of 25 μL. Reaction conditions: Pre-denaturation at 94°C for 2 min...

Embodiment 2

[0040] Reactogenicity and immunogenicity identification of embodiment 2S recombinant protein

[0041] Firstly, the above-mentioned bacterial liquid was ultrasonicated and then centrifuged to obtain inclusion body precipitation, and the inclusion body was denatured. The denatured supernatant was passed through His-Trap column affinity chromatography, and the liquid after passing through the column was collected, and then subjected to gradient dialysis with refolding buffer, and finally Collect the supernatant. After performing SDS-PAGE electrophoresis on the purified recombinant fusion protein, the electrophoresis results are as follows: image 3 As shown, electrotransfer to nitrocellulose membrane for Western blot identification, such as Figure 4 As shown, the results show that the feline coronavirus S recombinant protein of the present disclosure can react with cat serum and has biological reactogenicity. The purified and identified recombinant protein was dialyzed with PB...

Embodiment 3

[0042] Embodiment 3 is used to detect the development of the indirect ELISA kit of cat coronavirus antibody

[0043] 1. Determination of the optimal coating concentration of the antigen and the optimal concentration of the secondary antibody

[0044] (1) Dissolve the purified protein in the coating solution at an appropriate concentration, and start from left to right for doubling dilution (8μg / ml, 4μg / ml, 2μg / ml, 1μg / ml, 0.5μg / ml ml, 0.25 μg / ml). The antigen was added to the ELISA plate at 50 μl per well for overnight coating at 4°C.

[0045] (2) Take out the coated ELISA plate, pour off the liquid in the plate, and empty it, wash it with PBST (200μl) for 3 times, each time for 5min, use 5% skimmed milk to press 100μl / well, at 37℃ Closed for 2 hours.

[0046] (3) Take out the ELISA plate that has been blocked, pour off the liquid in the plate, and empty it, wash it with PBST (200μl) for 3 times, each time for 5min, and use 50μl / well of positive serum (primary antibody) at ...

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Abstract

The invention provides an indirect ELISA kit for detecting a feline coronavirus antibody. The kit contains a coating antigen S recombinant protein; the amino acid sequence of the S recombinant protein is as shown in SEQ ID NO: 1. The invention also provides application of the kit in detection of the feline coronavirus antibody. According to the indirect ELISA kit for detecting the feline coronavirus antibody of the invention, the ELISA detection kit for the feline coronavirus antibody is developed on the basis of an indirect ELISA principle. A conservative feline coronavirus S2 protein with strong antigenicity is screened out to amplify and clone a gene; a prokaryotic expression vector is constructed, namelya prokaryotic expression fusion protein; immunogenicity analysis is carried out through Western blotting, the S recombinant protein is purified into a coating antigen; and finally the indirect ELISA kit with relatively high sensitivity, strong specificity and good stability can be obtained.

Description

technical field [0001] The disclosure relates to the technical field of antibody detection, in particular to an indirect ELISA kit for detecting feline coronavirus antibody. Background technique [0002] Feline coronavirus (FCoV) is a non-segmented, single-stranded positive-sense RNA virus with a genome size of about 30kb, belonging to Nidovi-rale, Coronaviridae, Alphacoronavirus ). FCoV has 11 open reading frames (ORFs), encoding 4 structural proteins: spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), and 7 nonstructural proteins Proteins: accessory proteins 3a, 3b, 3c, 7a, 7b, and replicase 1a and 1b. The coronavirus S protein is 12-24nm in length, dome-shaped, arranged like a crown, and is the largest structural protein on the surface of the virus. The S protein plays an important biological role in the process of invading host cells through membrane fusion after the combination of virus particles and cell surface receptors and me...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/58G01N33/543C07K14/165C12N15/50C12N15/70
CPCG01N33/56983G01N33/581G01N33/54306C07K14/005C12N15/70G01N2333/165G01N2469/20Y02A50/30
Inventor 林炜明董波章高强陈雪菲张晓东李成钰魏兰
Owner LONGYAN UNIV
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