African swine fever virus truncated protein and application thereof in preparation of ELISA detection kit

An African swine fever virus and detection kit technology, applied in the biological field, can solve the problems of different detection effects of Elisa detection kits, and achieve the effects of strong clinical practicability, good specificity and simple operation steps

Active Publication Date: 2020-10-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For truncated proteins, even if different regions of the same protein are truncated, the detection antigens fo

Method used

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  • African swine fever virus truncated protein and application thereof in preparation of ELISA detection kit
  • African swine fever virus truncated protein and application thereof in preparation of ELISA detection kit
  • African swine fever virus truncated protein and application thereof in preparation of ELISA detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Obtaining of African swine fever virus truncated protein:

[0022] 1. Obtaining and identification of recombinant plasmids

[0023] The p54 gene sequence of the African swine fever Benin 97 / 1 strain (GenBank accession number: AM712239.1) was truncated and codon optimized, and then entrusted to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. to synthesize the p54 gene (the nucleotide sequence is shown in SEQ ID NO.2), and at the same time, the synthetic p54 gene was embedded into the pET32a vector to obtain the recombinant plasmid pET32a-p54. The recombinant plasmid pET32a-p54 was transformed into E.coliBL21(DE3) competent to obtain the recombinant strain pET32a-p54 / BL21(DE3). Randomly pick 3 single colonies and add each into 100 μl ultrapure water, boil for 10 minutes, take the supernatant as a template, and carry out PCR amplification according to the following reaction system and amplification procedure.

[0024] Primer sequence:

[0025] Upstream primer P1: 5'-GGCTGA...

Embodiment 2

[0036] Construction of African swine fever virus truncated protein indirect ELISA detection kit:

[0037] 1. Preparation of positive control:

[0038] Healthy negative pigs aged 7 to 8 weeks were selected, and the serum was required to be negative by the American Biostone African swine fever virus indirect ELISA antibody detection kit, and the nucleic acids of ASFV, CSFV, PRV, PPV, and JEV were detected by PCR or RT-PCR. feminine. The truncated p54 recombinant protein obtained in the present invention was mixed with an adjuvant at a ratio of 1:1, and 1 ml / head (protein content: 1.0 mg) was intramuscularly injected into the neck, and immunized twice in a row with an interval of 14 days between each time. 14 days after the second immunization, the blood test was started, and the serum ELISA titer was not lower than 1:8 with the American Biostone African Swine Fever Virus Indirect ELISA Antibody Detection Kit. Collect blood, centrifuge at 4000r / min for 10 minutes after the bloo...

Embodiment 3

[0053] The method of using the indirect ELISA detection kit prepared by African swine fever virus truncated protein:

[0054] Sample processing: take pig whole blood, centrifuge at 4000 rpm for 10 minutes after the blood coagulates, and collect the supernatant. Blood can also be collected, and the serum will be precipitated naturally after coagulation. The serum is required to be clear and free of hemolysis.

[0055] Washing solution preparation: Before use, take the concentrated washing solution out of the kit, equilibrate to room temperature (20-25°C), and shake to dissolve the precipitate (preferably heated in a 37°C water bath for 5-10 minutes), then Make a 20-fold dilution with distilled water (for example: use 30ml of 20-fold concentrated washing solution plus 570ml of distilled water for every two plates), mix well, and store the diluted washing solution at 2-8°C for 7 days.

[0056] Preliminary sample dilution: dilute the serum sample to be tested in the serum dilutio...

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Abstract

The invention belongs to the technical field of biology, and discloses an African swine fever virus truncated protein and application thereof in preparation of an ELISA detection kit, and the truncated protein is shown as SEQ ID NO. 1. When the indirect ELISA kit prepared by taking the protein as a coating antigen is used for detecting the African swine fever virus, the coincidence rate of the kitis equivalent, but the sensitivity of the kit is higher than that of a Spanish detection kit, the test time is shortened, and the operation steps are simpler. Therefore, the African swine fever virusindirect ELISA detection kit prepared from the truncated protein is very suitable for clinical detection of large samples and is suitable for large-scale popularization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a truncated protein of African swine fever virus and its application in preparing an ELISA detection kit. Background technique [0002] African swine fever (African Swine Fever, ASF) is caused by African swine fever virus (African SwineFeverVirus, ASFV), an acute, febrile and highly contagious zoonotic disease of pigs, the main clinical symptoms are skin congestion and cyanosis , Severe internal organ bleeding, high morbidity, high mortality, and short course of disease. The World Organization for Animal Health (OIE) lists it as a category A animal disease of legally notifiable animals, and this disease is regulated as a first-class animal infectious disease in my country. [0003] African swine fever virus is a double-stranded linear DNA virus with an icosahedral structure, a genome length of 170-190 kb, 151 open reading frames, and an envelope. African swine fever can...

Claims

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Application Information

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IPC IPC(8): C07K14/01C12N15/34C12N15/70G01N33/569G01N33/68C12R1/19
CPCC07K14/005C12N15/70C12N2710/12022C12N2800/22G01N33/56983G01N33/6854G01N2333/01G01N2469/20
Inventor 金梅林杨永张强赵亚孙小美吕长杰杨丽
Owner HUAZHONG AGRI UNIV
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