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Coding gene of duck costimulatory molecule CD40 and application of coding gene

A technology of co-stimulatory molecules and coding genes, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve the effect of broad application prospects

Inactive Publication Date: 2020-03-31
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since viruses are strictly obligate parasites, there is no strictly selective agent that can be effectively used in the prevention and treatment of animal viral diseases.

Method used

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  • Coding gene of duck costimulatory molecule CD40 and application of coding gene
  • Coding gene of duck costimulatory molecule CD40 and application of coding gene
  • Coding gene of duck costimulatory molecule CD40 and application of coding gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Cloning of the full-length sequence of duck co-stimulatory molecule CD40

[0032] 1.1 Cloning of the 3' end CDS of duck CD40

[0033] 1.1.1 Primer design

[0034] According to the incomplete predicted sequence of duck CD40 found in GenBank (GenBank ID: XM_021276409.1) and the predicted sequence of goose CD40 (GenBank ID: XM_013198462.1) with high homology to the predicted sequence of duck CD40, a pair of primers were designed, upstream Primer: 5'-CCAAAACCGAGCCGTGCTACCCC-3'; downstream primer: 5'-C GCCCTGCGCCCCTCACAGC-3'. After the primers were synthesized, they were dissolved in an appropriate amount of sterilized deionized water to make the final concentration 10 μM, and stored at -20°C for later use.

[0035] 1.1.2 Extraction of duck blood RNA

[0036] (1) Take an appropriate amount of duck blood and add it to a 1.5mLEP tube, add 1mL of Trizol Reagent, shake vigorously until it is completely lysed, and then let it stand at room temperature (25-27°C) for 1...

Embodiment 2

[0076]Preparation of embodiment 2 duck CD40 recombinant protein

[0077] 2.1 Construction of prokaryotic expression plasmid pET28a-CD40, induction of expression and optimization of expression conditions

[0078] 2.1.1 Primer design

[0079] According to the nucleotide sequence corresponding to the protein sequence encoded by duck CD40, a pair of primers were designed, the upstream primer: 5'-CGGAATTCATGCACTGCTCAGACACCAGC-3'; the downstream primer: 5'-CCGCTCGAGTCACAGCCCCTCCTGCTCC-3'. After the primers were synthesized, they were dissolved in an appropriate amount of sterilized deionized water to make the final concentration 10 μM, and stored at -20°C for later use.

[0080] 2.1.2 Construction and identification of prokaryotic expression plasmid pET28a-CD40

[0081] Using pMD19T-CD40 as a template, amplify the target fragment with the primers described in 2.1.1, and recover and purify it. Then the target fragment and pET28a(+) empty plasmid were subjected to double enzyme dig...

Embodiment 3

[0116] Example 3 Preparation and application of duck CD40 recombinant protein polyclonal antibody

[0117] 3.1 Preparation of polyclonal antibody against duck CD40 recombinant protein

[0118] 3.1.1 Animal immunization procedures

[0119] Using the purified duck CD40 recombinant protein as the antigen, mix it with an equal amount of Freund's complete adjuvant, shake vigorously to fully emulsify the antigen into a water-in-oil emulsion seedling. Twelve healthy female Kunming rats were taken, and each rat was intradermally injected with the aforementioned emulsion vaccine (50 μg of duck CD40 recombinant protein) for primary immunization. Two weeks after one immunization, the purified duck CD40 recombinant protein was mixed with an equal amount of Freund's incomplete adjuvant, vigorously shaken to emulsify the antigen fully into a water-in-oil emulsion vaccine, and then each mouse was subcutaneously injected with the emulsion vaccine (duck The amount of CD40 recombinant protein...

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Abstract

The invention discloses a coding gene of duck costimulatory molecule CD40 and an application of the coding gene, and belongs to the technical field of biology. The nucleotide sequence of the coding gene is shown in SEQ ID No:1, an protein encoded by the gene is composed of 171 amino acid residues, the amino acid sequence is shown in SEQ ID No:2, and the protein molecule forms an extracellular tumor necrosis factor receptor structural domain, a transmembrane structural domain and an intracellular structural domain, and is a 18.8kD I-type transmembrane glycoprotein. The CDS full sequence of theduck costimulatory molecule CD40 provided by the invention has greater practical significance in the development of related detection kits, and the antiviral function has broad application prospects in prevention and treatment of waterfowl diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a gene encoding duck costimulatory molecule CD40 and its application. Background technique [0002] Duck is a waterfowl with important economic value. During its growth and development, it is vulnerable to various diseases, among which viral damage is the most serious. The most common virus invading ducks at present has duck plague virus, duck hepatitis virus, duck Tembusu virus or the like. Since viruses are strictly obligate parasites, no strictly selective medicament can be effectively used in the prevention and treatment of animal viral diseases so far. In production practice, preventive measures such as eliminating the source of the virus, cutting off the transmission route of the virus, and developing and producing different vaccines are mainly used to control the harm of viral diseases. Among them, the development and production of vaccines targeting specific pathogens is th...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C12N1/21C07K14/705C07K16/28A61P31/14C12R1/19
CPCA61P31/14C07K14/70578C07K16/2878C12N15/70
Inventor 程安春张铷娟汪铭书
Owner SICHUAN AGRI UNIV
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