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Duck adenovirus type I Penton protein as well as preparation method and application thereof

An adenovirus and protein technology, applied in the field of protein engineering, can solve the problems of unsuitable chicken flock antibody monitoring and epidemiological investigation, economic losses of farmers and breeding enterprises, unachievable egg production of chicken flocks, etc. The effect of mutation rate, strong reactogenicity and low cost

Inactive Publication Date: 2020-09-25
ANYANG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the laying hens are infected with the EDS virus, the egg production of the flock cannot reach the peak period or the egg production drops sharply, which can reduce the egg production by 20% to 30%, which seriously affects the development of the laying hen industry. Breeding enterprises bring huge economic losses, and the harm is very serious
[0003] At present, DAV1 is distributed all over the world, which has brought serious economic losses to the poultry industry in many countries. ELLSA has the advantages of simplicity, speed, sensitivity, and specificity in detecting antibodies, and has been widely used in the production field. The current kit mainly uses the whole virus as the coated antigen, the process of purifying the virus particles is complicated, and there is a risk of spreading the virus, and it is not suitable for a series of difficult promotion and application problems such as the monitoring of the antibody level of a large number of chicken flocks and epidemiological investigation.

Method used

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  • Duck adenovirus type I Penton protein as well as preparation method and application thereof
  • Duck adenovirus type I Penton protein as well as preparation method and application thereof
  • Duck adenovirus type I Penton protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Amplification of Penton protein sequence and construction of prokaryotic expression plasmid pET-11a-Penton:

[0026] The whole gene is used to synthesize the Penton protein gene, the amino acid sequence listing of the Penton protein is SEQ.ID.No.1, the nucleotide sequence listing of the recombinant Penton protein gene is SEQ.ID.No.2, through restriction enzyme Insert the recombinant Penton protein gene into the pET-11a expression vector at the cutting sites NdeI and BamHI, and add 3 μL of the recombinant pET-11a-Penton plasmid, 0.25 μL of endonuclease Ⅰ, and 0.25 μL of endonuclease Ⅱ into a 25 μL centrifuge tube , 10×Buffer 1.0 μL, mix well, place in a low-temperature ligation instrument at 16°C for ligation reaction for 30 minutes, and obtain ligation products, which are double-digested and sequenced by restriction enzyme sites Nde I and BamHI The accuracy of the final expression vector was confirmed to obtain the prokaryotic expression plasmid pET-11a-Penton. figu...

Embodiment 2

[0035] This embodiment is the application of the Penton protein described in Example 1. The Penton protein is used for the indirect ELISA kit for detecting duck adenovirus type I antibody. Serum, positive control serum, enzyme-labeled secondary antibody, enzyme-labeled secondary antibody diluent, buffer, blocking solution, diluent, washing solution, color developing solution and stop solution, and the coated microtiter plate is coated with Penton protein antigen.

[0036] 1. Preparation of enzyme-labeled secondary antibody: Dilute rabbit anti-duck IgG (produced by Beijing Biolab Technology Co., Ltd.) with HRP conjugate stabilizer / diluent I (produced by Huzhou Yingchuang Biotechnology Co., Ltd.) 100 times and get.

[0037] 2. Preparation of coated enzyme-labeled secondary antibody diluent, buffer, blocking solution, sample diluent, and washing solution:

[0038] The enzyme-labeled secondary antibody diluent is a 0.01M phosphate buffered saline solution b containing pH 7.2 to ...

Embodiment 3

[0075] The duck adenovirus type I antibody indirect ELISA method of this example was used to detect 98 chicken serum samples submitted for clinical examination. Chicken serum detected 25 duck-type 1 adenovirus-positive sera, with a positive rate of 25%, indicating that the duck-type 1 adenovirus had a high infection rate in the chicken flocks submitted for inspection this time. Purified Penton protein was used as the package The indirect ELISA method established by the antigen has the characteristics of good specificity and high sensitivity.

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Abstract

The invention belongs to the field of protein engineering, and particularly relates to duck adenovirus type I Penton protein as well as a preparation method and application thereof. The inventor, by analysis, finds that the Penton protein has the advantages of hydrophilicity, no transmembrane structural domain, no signal peptide prediction and the like, and full-length expression is selected. A Penton target fragment is obtained through artificial synthesis after sequence optimization, and the expression mode of the protein is cell-free protein expression. Compared with truncated expression ofantigen dominant epitopes of traditional Penton protein, the artificially synthesized and expressed Penton recombinant protein is high in accuracy, the neutrality of the protein is guaranteed, the trouble of high mutation rate is avoided, and the application to the development of an antibody detection technology is better facilitated. In addition, an expression system has unique advantages of saving time and improving the total yield of soluble full-length protein. Furthermore, the expression system is low in cost, large in expression quantity and high in Western Blot detection reactogenicity.

Description

technical field [0001] The invention belongs to the field of protein engineering, in particular to a duck adenovirus type I Penton protein and its preparation method and application. Background technique [0002] Duck adenovirus type 1 (DAV1) is a type of adenovirus including Egg drop syndrome virus (EDSV) or EDSV-like, belonging to the genus Thymovirus of the family Adenoviridae. member. Its representative virus is Egg Drop Syndrome (EDSV), which is a viral infectious disease that reduces the egg production rate of laying hens or breeding hens. After the laying hens are infected with the EDS virus, the egg production of the flock cannot reach the peak period or the egg production drops sharply, which can reduce the egg production by 20% to 30%, which seriously affects the development of the laying hen industry. Breeding enterprises bring huge economic losses, and the harm is very serious. [0003] At present, DAV1 is distributed all over the world, which has brought seri...

Claims

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Application Information

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IPC IPC(8): C07K14/075C12N15/70G01N33/569C12R1/19
CPCC07K14/005C12N15/70G01N33/56983C12N2710/10122G01N2333/075G01N2469/20
Inventor 王雪平张孝俊梁秀丽
Owner ANYANG INST OF TECH
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