African swine fever virus antibody ELISA detection kit and preparation method thereof

An African swine fever virus and detection kit technology, which is applied in the field of immunoassays, can solve the problems of inability to give an accurate judgment of infection in time, time-consuming and other problems, and achieve the effects of improving accuracy, improving sensitivity and high detection sensitivity.

Active Publication Date: 2020-11-13
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the main diagnosis of African swine fever is based on virus gene detection, through RT-PCR and partial genome sequence analysis, which takes a long time and cannot give an accurate judgment of infection in time. Therefore, it is urgent to develop a clinically Fast, accurate and effective ELISA detection reagent products

Method used

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  • African swine fever virus antibody ELISA detection kit and preparation method thereof
  • African swine fever virus antibody ELISA detection kit and preparation method thereof
  • African swine fever virus antibody ELISA detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 Preparation of African swine fever virus Δp54 protein

[0034] 1. Construction of recombinant Escherichia coli engineering bacteria E.coli BL21 / pET-Δp54

[0035] The Δp54 protein gene sequence shown in SEQ ID NO.2 was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for full sequence synthesis and connected to the pET28a plasmid. The ligated plasmid was transformed into Escherichia coli BL21 (DE3), and a single clone was picked and cultured overnight in LB medium containing 100 μg / ml kanamycin. The plasmid was extracted and then sequenced and analyzed. The positive clone was the engineering bacterium E.coli BL21 / pET-Δp54.

[0036] 2. Expression of Δp54 protein

[0037] Inoculate 1ml of E.coli BL21 / pET-Δp54 strain into 100ml of LB liquid medium containing kanamycin, shake at 220 rpm at 37°C for 2 hours, lower the temperature to 28°C, and add the final concentration 0.5mmol / L IPTG solution induced culture for 12 hours. After the bacterial culture was com...

Embodiment 2

[0040] The preparation of embodiment 2 African swine fever virus p30 protein

[0041] 1. Construction of recombinant Escherichia coli engineering bacteria E.coli BL21 / pET-p30

[0042] The p30 protein gene sequence shown in SEQ ID NO.1 was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for full sequence synthesis, and connected to the pET28a plasmid. The ligated plasmid was transformed into Escherichia coli BL21 (DE3), and a single clone was picked and cultured overnight in LB medium containing 100 μg / ml kanamycin. The plasmid was extracted and then sequenced and analyzed. The positive clone was the engineering bacterium E.coli BL21 / pET-p30.

[0043] 2. Expression of p30 protein

[0044] Inoculate 1ml of E.coli BL21 / pET-p30 strain into 100ml of LB liquid medium containing kanamycin, shake at 220 rpm at 37°C for 2 hours, lower the temperature to 28°C, and add the final concentration 0.5mmol / L IPTG solution induced culture for 12 hours. After the bacterial culture was comp...

Embodiment 3

[0047] The preparation of embodiment 3 African swine fever virus ELISA antibody detection kit

[0048] 1. Microplate preparation

[0049] Enzyme plate coating antigen: the African swine fever virus Δp54 protein and p30 protein prepared in Example 1 and Example 2 were coated with the components corresponding to the original number as shown in Table 1 with carbonate buffer solution of pH 9.6 Prepare with the coating amount, coat the ELISA plate, seal the plate with a plate sealing film, and coat at 2-8°C for 16-24 hours. Discard the liquid in the well, wash once with washing solution, 300 μl / well, and pat dry on absorbent paper.

[0050] Table 1 Coating raw components and their contents corresponding to each kit

[0051]

[0052] Blocking: 150 μl of phosphate buffer (pH 7.4) containing 20% ​​newborn bovine serum was added to each well to block at 2-8° C. for 16-24 hours. Dry in an environment at 18-26°C and relative humidity below 30% for 3-6 hours;

[0053] Sample diluen...

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Abstract

The invention provides an African swine fever virus antibody ELISA (Enzyme Linked Immuno Sorbent Assay) detection kit. A support medium of the kit is coated with an African swine fever virus antigen,and the African swine fever virus antigen is African swine fever virus p30 and / or protein delta p54 protein. The African swine fever virus p30 protein is as shown in SEQ ID No. 1, and the African swine fever virus delta p54 protein is as shown in SEQ ID No. 2. The African swine fever virus antibody ELISA detection kit disclosed by the invention is good in specificity, sensitivity and repeatability, the sensitivity of the kit coated with the two antigens is equivalent to that of indirect immunofluorescence detection, antibody positive can be detected earlier at a low antibody level in the earlystage, a basis is provided for clinical swine herd infection conditions, and the kit plays an important role in preventing and controlling African swine fever virus infection.

Description

technical field [0001] The invention belongs to the field of immunoassays, and in particular relates to an African swine fever virus antibody ELISA kit and a preparation method thereof. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious infectious disease of pigs caused by African swine fever virus (ASFV). It is characterized by a short course of disease and a high fatality rate, which can be as high as 100%. The clinical symptoms and pathological changes are similar to acute swine fever, and it is easy to be misdiagnosed during diagnosis. [0003] The disease is listed as a first-class animal disease in my country, and belongs to the legally notifiable animal disease of the World Organization for Animal Health (OIE). my country reported the first case of African swine fever in 2018, and it spread rapidly to various provinces (regions). The number of live pigs in China accounts for 50% of the world, and the spread of ASFV in Chi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/533G01N33/569
CPCG01N33/533G01N33/535G01N33/56983
Inventor 田克恭孙杰王彦伟邓均华
Owner LUOYANG PULIKE WANTAI BIOTECH
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