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Infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof

A technology for bronchitis and chicken infectivity, which is applied in the field of immunological detection, can solve the problems of serological detection methods for non-structural protein detection antigens and other problems, and achieve the effects of high sensitivity, strong specificity and high coincidence rate.

Inactive Publication Date: 2016-07-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no reports of serological detection methods using non-structural proteins of IBV as antigens

Method used

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  • Infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof
  • Infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof
  • Infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof

Examples

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Effect test

preparation example Construction

[0050](1) Preparation of ELISA antigen: Utilize IBVnsp5-specific primers (IBVnsp5-F and IBVnsp5-R, the primer sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4), extracted from chicken embryo allantoic fluid infected with IBVSC021202 strain The nucleic acid was used as a template, and the IBVnsp5 gene was amplified by RT-PCR method, cloned into the prokaryotic expression vector pEGX-4T-1, transformed into Escherichia coli, and the recombinant bacteria carrying the IBVnsp5 gene were screened, and expressed in large quantities under the induction of IPTG and GST The fused nsp5 recombinant protein was purified by GSTrapFF affinity chromatography column to obtain high-purity IBVnsp5 recombinant protein and stored at -20°C for later use; the nucleotide sequence of the IBVnsp5 gene is shown in SEQ ID NO: 1; the gene encoding of IBVnsp5 The amino acid sequence is shown in SEQ ID NO:2.

[0051] (2) Preparation of IBVnsp5-coated enzyme-linked reaction plate:

[0052] After the conc...

Embodiment 1

[0060] [Example 1] Preparation of antigen

[0061] The expression host bacterium E.coliBL21(DE3) carrying the pGEX-4T-1-nsp5 vector was activated by LB liquid medium, and transferred to LB liquid medium containing ampicillin resistance at a ratio of 1:100 for mass cultivation and propagation . OD of bacterial solution 600nm When it reaches 0.4-0.6, add IPTG with a final concentration of 1mM to induce expression for 5h, then centrifuge at 6,000rpm for 10min at 4°C to collect the bacteria, and use GSTrapFF affinity chromatography column to purify the target protein according to the product manual. Protein samples obtained after elution were analyzed by SDS-PAGE. Such as figure 1 As shown, the purified recombinant protein GST-nsp5 has a molecular weight of 60KD and is of high purity. Purified GST-nsp5 was stored at -20°C for future use.

Embodiment 2

[0062] [Example 2] Preparation and screening of negative and positive serum

[0063] The negative and positive serum used in the present invention was provided by Zhejiang Tongdian Biotechnology Co., Ltd. The serum preparation process is illustrated below, but it does not mean that the realization of the present invention must start from the preparation of serum, nor does it mean that the method of serum preparation is limited to the content described.

[0064] (1) Preparation of IBVnsp5 negative and positive serum

[0065] The positive serum used in this kit can be purified by the recombinant GST-nsp5 protein in Example 1, after immunizing SPF chickens at a dose of 1mg / only three times, collecting and separating the immune chicken serum, using indirect immunofluorescence assay (IFA) After verifying that the serum contains high-titer anti-nsp5 antibodies, it is aliquoted and stored at -70°C as IBVnsp5 positive serum. Serum from non-immunized SPF chickens was tested negative ...

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Abstract

The invention relates to the field of immunological detection and aims at providing an infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof. The kit comprises a 96-hole ELISA plate coated with recombination expression IBV nsp5, an IgG enzyme-labeled antibody solution diluted with sample diluent, confining liquid, a scrubbing solution, the sample diluent, a positive control sample, a negative control sample, developing liquid and stop liquid. According to the kit, nsp5 is adopted as coating antigen, the kit can have the character of being high in coincidence rate with an import commercialization kit, and cost is greatly reduced. IBV nsp is taken as antigen to assemble the ELISA antibody detection kit for the first time in the industry. The kit has the advantages that using is convenient and quick, sensitivity is high and specificity is strong; a new effective detection means is clinically provided for IBV large-scale antibody monitoring, epidemiological investigation and IB prevention and control.

Description

technical field [0001] The invention belongs to the field of immunological detection methods, and in particular relates to an ELISA antibody detection kit using infectious bronchitis virus nsp5 protein as a coated antigen and application thereof. Background technique [0002] Chicken infectious bronchitis (Infectiousbronchitis, IB) is an acute, highly contagious infectious disease of chickens caused by infectious bronchitis virus (Infectiousbronchitis Virus, IBV). Characterized by dyspnea, nephritis and decreased egg quality, the disease is widespread all over the world and is one of the most serious respiratory infectious diseases in the poultry industry. The IBV genome is about 27.4kb long, encoding 4 structural proteins (S, M, N, E), 15 non-structural proteins (nsp2-nsp16) and 4 accessory proteins 3a, 3b, 5a and 5b. Due to the unique transcription mechanism of IBV RNA and the lack of correction system of RNase, IBV is easy to mutate, and complex tissue tropism, serotype ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 廖敏雷静周继勇颜焰金玉兰
Owner ZHEJIANG UNIV
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