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Indirect ELISA method for identifying Newcastle disease infection and immunization

A technology for Newcastle disease and Newcastle disease virus, applied in the biological field, can solve problems such as unreported NDV non-structural protein serological detection methods, etc.

Inactive Publication Date: 2018-08-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, ELISA differential diagnosis methods have been established using non-structural proteins of viruses such as foot-and-mouth disease, reproductive and respiratory syndrome, avian influenza, and chicken infectious bronchitis at home and abroad, but there is no report of using NDV non-structural proteins as detection antigens. Serological testing method

Method used

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  • Indirect ELISA method for identifying Newcastle disease infection and immunization
  • Indirect ELISA method for identifying Newcastle disease infection and immunization
  • Indirect ELISA method for identifying Newcastle disease infection and immunization

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0050] Embodiment 1. Prokaryotic expression and purification of recombinant protein Vc

[0051] First of all, the envelope antigen used in this ELISA detection method is Newcastle disease virus recombinant protein Vc, which is expressed in large quantities through the prokaryotic expression system of E. Obtained after purification by nickel ion affinity chromatography column. The specific operation is as follows:

[0052] Take the prokaryotic expression vector pET-32a-Vc constructed in our laboratory to transform Escherichia coli BL21 competent cells, culture at 37°C for 12-14 hours, pick a single positive colony for expansion culture, and then add the inducer IPTG with a final concentration of 1mM Induced massive expression of recombinant protein Vc fused with His tag.

[0053] After prokaryotic expression, the bacterial cell precipitate of the expressed bacteria was collected, washed once with PBS, then added an appropriate amount of PBS to shake and mix well, and the supe...

Embodiment 2

[0062] The establishment of embodiment 2.Vc-ELISA detection method

[0063] 2.1 Basic procedure of ELISA

[0064] (1) Antigen coating: Dilute the antigen (i.e. recombinant protein Vc) to the working concentration with antigen coating solution (i.e. carbonate buffer solution at pH 9.6), coat the microtiter plate at 100 μL / well, and statically at 4°C. Leave it overnight, wash the plate 5 times with PBST (that is, PBS containing 0.05% Tween-20), shake it off and pat it dry.

[0065] (2) Blocking: Add blocking solution in an amount of 200 μL / well, place in a 37°C incubator to block for 2 hours, wash the plate 5 times with PBST, shake it off and pat dry.

[0066] (3) Incubate the primary antibody: Add the diluted serum to be tested and the negative and positive control sera (both negative and positive sera are repeated twice) in an amount of 100 μL / well, incubate for 1 hour in a 37°C incubator, and wash with PBST. Shake it off and pat dry after boarding 5 times.

[0067] (4) Inc...

Embodiment 3

[0100] Example 3. Vc-ELISA method for differential diagnosis of NDV infection and immunity

[0101] 3.1 Animal experiment design and HI results

[0102] Order 30 3-week-old SPF chickens, divide them into 3 groups, including the experimental group, PBS negative control group and virulent control group, 10 in each group, and raise them in SPF chicken isolators. Among them, 10 chickens in the experimental group were immunized for 3 weeks and then challenged with the virulent virus, and then continued to observe for 3 weeks, during which blood was collected as planned and serum was prepared (see Table 2).

[0103] Table 2 Animal experiment scheme

[0104]

[0105] Hemagglutination inhibition (HI) test results (see image 3 and Figure 4 ) shows that the experimental animal is successfully immunized, and the HI antibody titer (ie HI titer) can reach 10log after 3 weeks of immunization 2 After 3 weeks of immunization and then attacking NDV virulence, the HI antibody titer dec...

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Abstract

The invention belongs to an immunological detection method in the biotechnical field, concretely relates to an application of a special carboxy-terminal domain of a non-structural protein V of a Newcastle disease virus in the identification of Newcastle disease virus infection and immunization, and more concretely provides a method for differentially diagnosing Newcastle disease inactivated vaccine immunization and wild-type infection. An Escherichia coli prokaryotic expression system expresses the recombinant protein having the specific carboxy-terminal domain of the V protein, and Newcastledisease inactivated vaccine immunization and wild-type infection are identified through the indirect ELISA method with the recombinant protein as an antigen. The method has a high specificity, and canquickly and effectively distinguish the Newcastle disease wild-type infection and inactivated vaccine immunity in order to effectively control the rapid spreading of the Newcastle disease virus.

Description

technical field [0001] The invention belongs to the immunological detection method in the field of biotechnology, in particular to the prokaryotic expression of the unique carboxy-terminal domain of the nonstructural protein V of Newcastle disease virus, and the indirect ELISA for differential diagnosis of Newcastle disease virus infection and inactivated vaccine immunization using the prokaryotic expression product method. Background technique [0002] Newcastle disease (ND) is an acute and highly contagious infectious disease caused by Newcastle disease virus (NDV) in chickens and a variety of poultry. loss. The World Organization for Animal Health (OIE) lists ND as a statutory reportable disease, and ND is designated as a first-class animal disease in my country. The "National Medium- and Long-term Animal Disease Prevention and Control Plan (2012-2020)" lists ND as one of the five priority prevention and control One of the animal diseases. [0003] In terms of virus tax...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558G01N21/31
CPCG01N21/31G01N33/558G01N33/56983G01N2333/125
Inventor 任涛于得水徐成刚谢鹏梁健鹏林秋燕廖明
Owner SOUTH CHINA AGRI UNIV