Duck herpesvirus type 1 ul7 truncated recombinant protein and its preparation method and application

A technology of recombinant protein and duck herpes, which is applied in the field of bioengineering, can solve the problems of difficult purification of virus antigens, time-consuming and labor-intensive operations, and difficulty in standardization, etc., and achieves the effect of being suitable for large-scale industrial production, facilitating the comparison of results, and simple preparation methods

Active Publication Date: 2016-06-08
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the previous ELISA method uses the whole virus as the coating antigen. The purity of the whole virus antigen is limited, the stability is poor, and it is difficult to standardize, which affects the accuracy of the test results. The operation is time-consuming and laborious, which further limits the large-scale application of this method. Therefore, The preparation method of a new type of antigen is particularly important for the establishment of a specific and sensitive detection method to prevent and control AHV-1 infection
Similarly, antigen detection methods include neutralization test, immunofluorescence, etc., but the detection reagents also involve highly specific antibodies, and the preparation of antibodies (whether monoclonal or polyclonal antibodies) also involves the difficulty of purifying viral antigens

Method used

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  • Duck herpesvirus type 1 ul7 truncated recombinant protein and its preparation method and application
  • Duck herpesvirus type 1 ul7 truncated recombinant protein and its preparation method and application
  • Duck herpesvirus type 1 ul7 truncated recombinant protein and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1, the preparation of AHV-1UL7M recombinant protein

[0033] The amino acid sequence of the AHV-1UL7M recombinant protein to be prepared by the present invention is shown in SEQIDNo.2 (that is, the accession number is the AHV-1UL7 amino acid sequence from the 1st to the 241st amino acid sequence of AFC61876), and the nucleotide sequence of its encoding gene is as shown in SEQIDNo. .1 (that is, the accession number is the 1st to 723rd nucleotide sequence of the AHV-1UL7 gene of FJ222445).

[0034] 1. Cloning of AHV-1UL7M gene

[0035] 1. Design of primers

[0036] Design the following primers according to the AHV-1UL7M gene sequence: Upstream primer AHV-1-UL7MF: 5'- ggatcc atgga-tatgaaccagagc-3' (SEQ ID NO.3, the underlined part is the BamHI restriction site); downstream primer AHV-1-UL7MR: 5'- ctcgag tattgcatgattaggtag-3' (SEQ ID NO.4, the underlined part is the XhoI restriction site).

[0037] The above primers were synthesized by entrusting Dalian Bao...

Embodiment 2

[0060] Example 2, Preparation and Purification of AHV-1UL7M Recombinant Protein Polyclonal Antibody

[0061] 1. Preparation of polyclonal antiserum for animal immunization and AHV-1UL7M recombinant protein

[0062] The purified AHV-1 UL7M recombinant protein was used as antigen, mixed with an equal amount of Freund's complete adjuvant (purchased from Sigma), and vigorously shaken to fully emulsify the antigen to obtain water-in-oil emulsion vaccine A. Take 4 healthy male rabbits, collect 2 mL of blood from the ear artery, separate the serum as a negative control, and then inject emulsion vaccine A (AHV-1UL7M recombinant protein content is 0.6 mg) into each rabbit for primary immunization; two weeks after the primary immunization, Mix the purified AHV-1UL7M recombinant protein with an equal amount of Freund's incomplete adjuvant (mixed with lanolin and liquid paraffin at a volume ratio of 1:4), shake vigorously to fully emulsify the antigen, and obtain a water-in-oil emulsion ...

Embodiment 3

[0067] Embodiment 3, AHV-1UL7M recombinant protein detection AHV-1 antibody

[0068] Take the IPTG-induced expression product of the BL21pLysS engineering bacterium containing the recombinant expression plasmid pET32a-UL7M for SDS-PAGE electrophoresis, and set the IPTG-induced expression product of the BL21pLysS strain transformed with the empty vector pET-32a (+) as a control; after the electrophoresis, the protein The sample was transferred from the PAGE gel to the PVDF membrane by wet transfer method, and the obtained PVDF membrane was placed in a plate, and 10 mL of TBST containing 3% BSA was added, and the membrane was blocked by shaking gently at 37°C for 1 hour, and the membrane was washed 3 times with TBST, and then Add rabbit anti-AHV-1 serum or normal rabbit serum diluted with 0.5% BSA-containing TBST as the primary antibody, incubate overnight at 4°C, discard the primary antibody, wash the membrane three times with TBST, and then add 10 mL of HRP-labeled goat anti-r...

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Abstract

The invention discloses an anatid herpesvirus 1 (AHV-1) UL7 truncated recombinant protein shown in SEQ ID No.2, as well as a code gene, a recombinant expression vector and engineering bacteria of the AHV-1 UL7 truncated recombinant protein, and a method for preparing the truncated recombinant protein and a polyclonal antibody of the truncated recombinant protein from the engineering bacteria. The truncated recombinant protein is similar to a natural AHV-1 UL7 protein in immunoreactivity, can be specifically bound with an AHV-1 antibody to be served as a reagent for detecting the AHV-1 antibody. As the truncated recombinant protein is not a complete bacteria, the risk of poison diffusion can be avoided during detection; the preparation method is simple, and suitable for industrialized mass production; product standardization can be realized; comparison among results of different laboratories can be facilitated; the polyclonal antibody of the truncated recombinant protein can serve as a reagent for detecting an AHV-1 antigen, is high in specificity, cannot react with duck hepatitis virus and new type gosling enteritis virus in a crossed manner, and can serve as reagent application for positioning and distributing the UL7 protein in an AHV-1 infected host cell.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a virus truncated recombinant protein and its preparation method and application. Background technique [0002] Anatid Herpesvirus 1 (AHV-1) belongs to Herpesviridae, α-Herpesvirinae, and Marekvirus genus, which can cause an acute, febrile, contact-infected infection in waterfowl such as ducks, geese, and swans. Bloody infectious disease, the clinical manifestations of sick ducks are depression, high fever, ataxia, diarrhea, tearing and swelling of the head and neck of some sick ducks. The main clinical feature is the process of acute sepsis. The main habitat of wild waterfowl spreads rapidly, and the morbidity and mortality can be as high as 90%. It often causes serious economic losses and seriously threatens the survival and development of my country's waterfowl breeding industry. AHV-1 antigen or antibody detection is critical to its prevention and treatment. The main met...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/03C07K16/08C12N15/38C12N15/70C12N1/21C12P21/02G01N33/68G01N33/569
CPCC07K14/005C07K16/085C12N15/70C12N2710/16322G01N33/56994G01N33/6893
Inventor 汪铭书程安春黄杰贾仁勇杨乔孙昆峰陈舜刘马蜂朱德康陈孝跃
Owner SICHUAN AGRI UNIV
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