Porcine circovirus type 4 ELISA antibody detection kit, application and method for detecting porcine circovirus type 4 antibody

A porcine circovirus and antibody detection technology, applied in the biological field, can solve problems such as threats to the pig industry, and achieve strong specificity and sensitivity, accurate detection and/or identification

Pending Publication Date: 2021-02-09
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the epidemic intensity of porcine circovirus type 4 and the continuous strengthening and expansion of the epidemic area,

Method used

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  • Porcine circovirus type 4 ELISA antibody detection kit, application and method for detecting porcine circovirus type 4 antibody
  • Porcine circovirus type 4 ELISA antibody detection kit, application and method for detecting porcine circovirus type 4 antibody
  • Porcine circovirus type 4 ELISA antibody detection kit, application and method for detecting porcine circovirus type 4 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Preparation and purification of porcine circovirus type 4 Cap protein of embodiment 1

[0087] The present embodiment provides the preparation process of porcine circovirus type 4 Cap protein, comprising the following steps:

[0088] A1. After codon optimization, the nucleotide sequence of the porcine circovirus type 4 Cap gene obtained is shown in SEQ ID NO.2, and is connected on the expression vector pET-28a, obtains the recombinant vector and double enzyme digestion identification ( figure 1 );

[0089] A2. Transform the recombinant vector into the recipient bacterium BL21(DE3) to obtain the recombinant bacterium;

[0090] A3. Pick a positive single colony and culture it in the liquid medium until the OD600 is about 0.8, add IPTG to make the final concentration 0.5mM, induce at 20°C for 12-16 hours, and centrifuge to collect the precipitate;

[0091] A4. Add 1×PBS containing protease inhibitors to resuspend the pellet, sonicate, and centrifuge to get the supernatan...

Embodiment 2

[0097] Example 2 ELISA detection of porcine circovirus type 4 antibody

[0098] The present embodiment provides a kind of ELISA detection process of porcine circovirus type 4 antibody, comprising the following steps:

[0099] B1. Antigen coating: Dilute the purified recombinant protein with the coating solution to a final concentration of 0.625 μg / mL, add it to the microtiter plate, 100 μL / well, and coat overnight at 4°C;

[0100] B2. Washing: Wash the microtiter plate 3 times with PBST, let stand for 5 minutes each time, and pat dry with absorbent paper for the last time;

[0101] B3. Blocking: add blocking solution containing 0.25% gelatin PBST, 150 μL / well, block at 37°C for 1 hour;

[0102] B4. Washing: Wash the microtiter plate 3 times with PBST, pat dry on absorbent paper, and let stand for 5 minutes each time;

[0103] B5. Primary antibody incubation: Serum samples were diluted 200 times and added to the microtiter plate, and negative and positive controls were added ...

experiment example

[0112] In order to verify the beneficial effect of the different ELISA conditions that the present invention selects, carry out following experiment:

[0113] 1. Determination of the optimal dilution concentration of antigen and primary antibody

[0114] The purified recombinant protein was diluted with coating solution, coated with 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 100 μL / well, overnight at 4 °C; washed 3 times with PBST, each Add blocking solution for 1 hour at 37°C; after washing, dilute negative and positive sera at 1:100, 1:200, 1:400, and 1:800, add 100 μL / well, and incubate at 37°C for 90 minutes; wash Afterwards, add 1:10000 diluted HRP-rabbit anti-pig IgG, 100 μL / well, incubate at 37°C for 45 minutes; wash, pat dry, add 100 μL TMB color developing solution to each well, and protect from light at 37°C for 6 minutes; add 2M sulfuric acid, 50 μL / well, terminate the reaction, and read with a microplate reader. According to Table 1, when the coating concentra...

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Abstract

The invention provides a porcine circovirus type 4 ELISA antibody detection kit, application and a method for detecting a porcine circovirus type 4 antibody, and relates to the technical field of biology. The porcine circovirus type 4 ELISA antigen detection kit provided by the invention comprises an elisa plate coated with porcine circovirus type 4 Cap protein. The ELISA antibody detection kit isestablished by using the circovirus type 4 Cap protein as an antigen, and the Cap protein as a circovirus conservative protein has good antigenicity and can be specifically combined with a circovirustype 4 antibody in a serum sample, so that the kit provided by the invention has very strong specificity and sensitivity to the circovirus type 4.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a porcine circovirus type 4 ELISA antibody detection kit, its application and a method for detecting porcine circovirus type 4 antibody. Background technique [0002] Circoviruses that have been identified in pigs include porcine circovirus type 1 (PCV1), circovirus type 2 (PCV2), and circovirus type 3 (PCV3). However, in April 2019, circovirus type 4 (PCV4) was found in several clinically ill pigs in Hunan Province, my country. In addition, there are research reports that PCV4 was also found in certain pig farms in Ningxia, Henan and Shanxi provinces of my country. The genome size of the virus is 1770 nucleotides (nt), and it has the highest genotype homology (66.9%) with mink circovirus, and 43.2%-51.5% homology with other PCV genomes. It contains two main genes, Rep gene (891nt) and Cap gene (687nt). At present, PCV4 has been found in many provinces in my country, and it has be...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569G01N33/58G01N33/543
CPCG01N33/54393G01N33/56983G01N33/581G01N33/6854G01N2333/01G01N2469/20
Inventor 涂忠忠涂长春何彪杜海莹冯烨
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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