Duck plague virus ul15 gene exoni recombinant protein and its preparation method and application

A duck plague virus and recombinant protein technology, which is applied in the field of biochemistry, can solve the problems of easy to disperse, lack of generalization, and unsatisfactory purity, and achieves the effect of strong specificity

Active Publication Date: 2017-10-31
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the methods for detecting duck plague virus antibodies are based on the whole virus of duck plague virus as an antigen detection method, but this method is easy to disperse the virus, and the preparation of the antigen is relatively complicated and the purity is not ideal, which makes this method not popular. sex

Method used

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  • Duck plague virus ul15 gene exoni recombinant protein and its preparation method and application
  • Duck plague virus ul15 gene exoni recombinant protein and its preparation method and application
  • Duck plague virus ul15 gene exoni recombinant protein and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, the construction of duck plague virus UL15 gene exonI recombinant expression plasmid

[0034] (1) Strains, plasmids and strains

[0035] Plasmid pMD18-T was purchased from Dalian Bao Biological Engineering Co., Ltd.; prokaryotic expression plasmid pPAL7 was purchased from Bio-Rad Company (article number: 156-3002); Escherichia coli DH5α, Escherichia coli Rossetta and duck plague The virus (DPV) CHv virulent strain was provided by the Poultry Disease Research Center of Sichuan Agricultural University.

[0036] (2) Test animals

[0037] The 10-day-old duck embryo was negative for DPV and DPV antibody of the duck embryo.

[0038] (3) Cloning of duck plague virus UL15 gene exonI

[0039] Design a pair of primers according to the DNA sequence of duck plague virus UL15 gene exon I, the upstream primer is 5'- ggatcc atcagtggcgtattatgtg-3' (SEQ ID NO.1), the underline indicates the BamH I restriction site, a total of 25bp, the 5' end is located at the 45th po...

Embodiment 2

[0056] Embodiment 2, the induced expression of duck plague virus UL15 gene exonI

[0057] (1) Extraction of recombinant plasmid pPAL7-UL15exonI

[0058] Pick the DH5α strain identified to contain the positive recombinant plasmid pPAL7-UL15exonI and inoculate it on the LB agar plate containing Amp, culture overnight at 37°C, inoculate a single colony on the LB liquid medium the next day, and culture it vigorously for 10~ After 16 hours, the bacterial liquid was collected by centrifugation, and the recombinant plasmid was extracted and purified according to the instructions of the U1traPureTM plasmid DNA mini-extraction kit.

[0059] (2) Transformation expression bacteria with recombinant plasmid pPAL7-UL15exonI

[0060] Escherichia coli Rossetta competent cells were prepared by the calcium chloride method, and the recombinant plasmid pPAL7-UL15exonI extracted above was transformed into the expression host bacteria Escherichia coli Rossetta, and positive clones were screened wi...

Embodiment 3

[0072] Embodiment 3, duck plague virus UL15 gene exonI recombinant protein detects duck plague virus antibody

[0073] (1) Extraction of duck anti-duck plague virus IgG

[0074] Take 5ml of duck anti-duck plague virus serum and place it in a 50ml centrifuge tube, add 5ml of normal saline, and then add (NH 4 ) 2 SO 4 Saturated solution 2.5ml, make it 20% (NH 4 ) 2 SO 4 solution, stirring while adding, after fully mixing, let it stand for 30min; then centrifuge at 4°C, 3000r / min for 20min, discard the precipitate, and then add (NH 4 ) 2 SO 4 Solution 7.5ml, make it 50% (NH 4 ) SO 4 After the solution was fully mixed, let it stand for 30 minutes; then centrifuged at 4°C, 3000r / min for 20 minutes, discarded the supernatant, added 5ml of normal saline to the precipitate to dissolve it, and then added saturated (NH 4 ) 2 SO 4 solution 2.5ml, making it 33.3% (NH 4 ) 2 SO 4 Mix the solution thoroughly and let it stand for 30 minutes; then centrifuge at 4°C, 3000r / min fo...

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Abstract

The invention discloses a duck plague virus UL15 gene exonI recombinant protein as well as a preparation method and application thereof. The amino acid sequence of the duck plague virus UL15 gene exonI recombinant protein is as shown in SEQ ID NO.4. The preparation method comprises the following steps: overnight culturing escherichia coli Rossetta containing a recombinant expression vector in an Amp-containing LB liquid culture medium; and transferring a bacterium solution into a fresh Amp-containing LB liquid culture medium, culturing until OD600 is 0.4, and adding IPTG for induced culture to obtain a culture solution of the duck plague virus UL15 gene exonI recombinant protein. The recombinant protein has immunocompetence, can be specifically combined with the antibody of duck plague virus, can be used as a detection reagent of duck plague virus antibody or used as an antigen for preparing a polyclonal antibody, so that a foundation is laid for detection of duck plague virus.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a duck plague virus UL15 gene exonI recombinant protein, and also relates to a preparation method and application of the recombinant protein. Background technique [0002] Duck plague (Duck Plague, DP) is an acute, febrile, contact-infected septicemia infectious disease of waterfowl such as ducks, geese and swans caused by duck plague virus (DPV) in the Herpesviridae family. . The disease can lead to a decrease in egg production and death of commercial waterfowl, and it also has a different lethality rate to wild waterfowl. In recent years, with the intensive and large-scale development of the duck industry, duck plague, as one of the most serious contagious diseases threatening the duck industry, has caused huge economic losses. [0003] Clinical and laboratory tests have confirmed that attenuated duck plague virus vaccine is an effective biological agent for the preven...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/03C12N15/38C12P21/02
CPCC07K14/005C12N2710/16022
Inventor 杨乔程安春汪铭书孙昆峰贾仁勇朱德康陈舜刘马峰
Owner SICHUAN AGRI UNIV
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