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Indirect enzyme linked immunosorbent assay (ELISA) detection method for European- and American-type porcine reproductive and respiratory syndrome virus (PRRSV) antibodies

A respiratory syndrome and detection method technology, applied in the field of indirect ELISA detection, can solve the problems of not being able to meet the diagnosis of PRRS infection, heavy workload, and long time consumption

Active Publication Date: 2011-09-07
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these diagnostic methods require special instruments and equipment, take a long time, and have a large workload, which can no longer meet the current needs of diagnosing PRRS infection, especially not suitable for large-scale clinical sample testing and epidemiological investigations.

Method used

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  • Indirect enzyme linked immunosorbent assay (ELISA) detection method for European- and American-type porcine reproductive and respiratory syndrome virus (PRRSV) antibodies
  • Indirect enzyme linked immunosorbent assay (ELISA) detection method for European- and American-type porcine reproductive and respiratory syndrome virus (PRRSV) antibodies
  • Indirect enzyme linked immunosorbent assay (ELISA) detection method for European- and American-type porcine reproductive and respiratory syndrome virus (PRRSV) antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Computer Screening of Antigenic Epitopes

[0034] Using computer software (DNAMAN, DNAStar, etc.) to carry out homology analysis of nucleotide and amino acid levels on porcine reproductive and respiratory syndrome virus nonstructural protein Nsp7 gene. The amino acid and nucleotide homology of American strains JXA1 (EF112445) and VR2332 (AY150564) were 89.8% and 90.0%, respectively; the amino acid and nucleotide homology between European strains were both 90% Above; American strains and European strains in terms of amino acid and nucleotide homology were: 50.3%, 47%.

[0035] Table 1 Comparison of Homology of Porcine Reproductive and Respiratory Syndrome Virus Nsp7 Gene at Nucleotide Level

[0036]

[0037] Table 2 Homology comparison of porcine reproductive and respiratory syndrome virus Nsp7 gene at amino acid level

[0038]

[0039] The screening of antigenic epitopes was mainly carried out by DNAStar (Madison, Wisconsin, USA) and DNAMAN software to analyze t...

Embodiment 2

[0041] Amplification of target gene and construction of expression vector

[0042] 2.1 Experimental reagents

[0043] DNA Polymerase, restriction enzyme BamH I, restriction enzyme EcoR I, restriction enzyme Hind III, enzyme digestion buffer 10×M Buffer, enzyme digestion buffer 10×H Buffer and DL2000 Marker were purchased from TaKaRa Company; Taq DNA polymerase, AMV reverse transcriptase, RNase inhibitors, and dNTPs were purchased from Promega; E.Z.N. Gel Extraction Kit, DNA glue recovery kit, purchased from OMEGA company;

[0044] High-efficiency Escherichia coli competent cells Trans-T1 were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0045] Primer synthesis and sequence sequencing were completed by Shanghai Yingjun Bioengineering Technology Service Co., Ltd.;

[0046] Other biochemical reagents are imported subpackages or domestic analytical pure.

[0047] 2.2 Experimental Instruments

[0048] TGRANDIENT PCR instrument: purchased from HYBAID company;...

Embodiment 3

[0067] Induced expression of recombinant expression bacteria (containing plasmids pET-32a-JXA1-ΔNsp7, pET-32a-LV-ΔNsp7)

[0068] 3.1 Experimental reagents

[0069] Blue Plus Protein Marker (16kDa-94kDa), Escherichia coli competent cells Transetta (DE3) for expression were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0070] 3.2 Experimental Instruments

[0071] HZQ-Q full temperature oscillator: purchased from Harbin Donglian Electronic Technology Development Co., Ltd.;

[0072] Mini-PROTEAN protein vertical electrophoresis instrument: purchased from BIORAD company;

[0073] VCX-400 ultrasonic cracker: purchased from Amersham pHarmacia Biotech;

[0074] 3.3 Experimental process

[0075] Streak the above-mentioned BL21(DE3) strain containing recombinant plasmids pET-32a-JXA1-ΔNsp7 and pET-32a-LV-ΔNsp7 on the LB plate containing ampicillin (Amp), cultivate overnight at 37°C, and pick a single colony at random , with 100ml of LB containing Amp antibiotics in a sh...

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Abstract

The invention provides a serological method for identifying European- and American-type porcine reproductive and respiratory syndrome virus (PRRSV) antibodies. According to the method, a prokaryotic recombinant expression carrier pET-32a-JXA1-[delta]Nsp7 of an American-type PRRSV Nsp7 targeted at the amino acid sequences from a position 1 to 23 to a position 59 to 259 of American-type PRRSV nonstructural protein Nsp7 protein is established; and a prokaryotic recombinant expression carrier pET-32a-LV-[delta]Nsp7 of a European-type PRRSV nonstructural protein Nsp7 targeted at the amino acid sequences from a position 1 to 23 to a position 59 to 269 of European-type PRRSV Nsp7 protein epitope is established. Recombinant plasmid is successfully expressed in colon bacillus BL21 (DE3). The indirect ELISA diagnosis kit for detecting the American-type PRRSV antibody is prepared by taking a nickel column affinity-purified American-type Nsp7 protein as a coating antigen, and the indirect ELISA method for detecting the European-type PRRSV antibody is optimized by using purified European-type Nsp7 protein as a coating antigen. The two kits are can be used for genotyping and detecting PRRSV antibody.

Description

technical field [0001] The invention relates to the animal disease diagnosis technology in the field of biotechnology. Using the similar antigenic epitope 24aa-58aa deleted Nsp7 gene expression protein of PRRSV JXA1 strain and LV strain as an antigen, a method for distinguishing European and American type pigs from breeding and breeding is established. Indirect ELISA detection method for respiratory syndrome virus antibody. Background technique [0002] According to the eighth report of the International Committee on Taxonomy of Viruses (ICTV) in 2005, porcine reproductive and respiratory syndrome virus (Procine reproductive and respiratory syndrome virus, PRRSV) belongs to Arteriviridae in classification Arterivirus. According to the sequence analysis of each isolate and the results of serological tests, PRRSV can be divided into two types: European type (the representative strain is LV) and North American type (the representative strain is VR2332), the former is mainly pr...

Claims

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Application Information

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IPC IPC(8): G01N33/569C12N15/40C12N15/11C12N15/70C07K14/08
Inventor 田克恭邱鹏倪建强顾小雪曲萍翟新验陈西钊
Owner CHINA ANIMAL DISEASE CONTROL CENT
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