Inactivated vaccine strain CH-GD-12-2014 against duck adenovirus type 3

A technology of inactivated vaccines and adenoviruses, applied in the direction of vaccines, viruses, viral peptides, etc., can solve the problems of lack of sequence and endanger the development of poultry industry, and achieve the effect of wide application value

Inactive Publication Date: 2018-07-27
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2011, scientists such as Harrach once again proved that the virus is a new virus and proposed to name it DAdV-2, but due to lack of sequence, the proposal was not adopted by ICTV
In recent years, the prevalence of duck adenovirus has shown an outbreak trend, especially the emergence of new muta

Method used

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  • Inactivated vaccine strain CH-GD-12-2014 against duck adenovirus type 3
  • Inactivated vaccine strain CH-GD-12-2014 against duck adenovirus type 3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Isolation and Identification of Duck Adenovirus Type 3 CH-GD-12-2014 Strain

[0020] CH-GD-12-2014 isolated and identified sick duck livers collected from duck farms in 2014. The specific process is as follows:

[0021] a. Virus isolation and culture

[0022] a.1 Culture of duck embryo fibroblasts

[0023] Aseptically collect 11-day-old non-immune fertilized duck embryos, remove the head, wings, claws and internal organs of the duck embryos, wash 3 times with PBS solution, and cut them to less than 3mm with sterilized scissors 3 After the tissue block, add PBS solution and shake gently, then add 0.25% trypsin, and digest in a water bath at 37°C until the liquid becomes uniform, then add serum to terminate the digestion. This process generally takes 20-30min; After filtering with a 100-mesh cell sieve, collect the filtrate in a 5mL centrifuge tube, centrifuge at 8000 rpm for 5min, remove the supernatant, add DMEM, and dilute to 5×10 5 Individuals / mL (contai...

Embodiment 2

[0039] Example two: research on duck adenovirus type 3 CH-GD-12-2014 inactivated vaccine

[0040] a. Physicochemical detection of virus

[0041] The results of physical and chemical tests show that CH-GD-12-2014 is resistant to acid and alkali, and is not sensitive to temperature. Ethyl ether, acetone, and chloroform have little effect on CH-GD-12-2014. Ultraviolet radiation and formaldehyde can make CH- GD-12-2014 inactivation;

[0042] b. Determination of virus content

[0043] Determination of TCID of CH-GD-12-2014 strain virus 50 , the virus was diluted 10 times with sterilized normal saline, and the diluted virus was inoculated into a single layer of DEF cells on 96 wells, and each dilution was inoculated in a vertical row with a total of 8 wells, each well was 0.1m1, and a control group was set ; After 2 hours of incubation, suck out the virus solution and add 100 μL of DMEM maintenance solution containing 2% serum to each well, at 37°C, CO 2 After culturing in a con...

Embodiment 3

[0050] Embodiment three CH-GD-12-2014 inactivated vaccine preparation

[0051] a. Preparation of CH-GD-12-2014 virus liquid

[0052] After thawing the DEF cell virus solution collected in the sixth generation, centrifuge at 10,000 rpm for 10 min, filter it with a microporous membrane with a pore size of 0.22 μm, and inoculate it into 200 75 cm cell culture flasks with 80%-90% monolayer DEF cells, 1ml per bottle, after inoculation, place at 37°C, CO 2 After culturing in the incubator for 3 hours, add DMEM maintenance solution (containing 2% serum) and incubate for 4-5 days. After the virus solution has been frozen three times and thawed three times, centrifuge at 10,000 rpm for 10 minutes to take the supernatant and filter it with a microporous membrane with a pore size of 0.22um. Collect cell venom, take samples for inspection, and store at -20°C for later use;

[0053] b. CH-GD-12-2014 venom inspection

[0054] The harvested virus liquid was subjected to sterility test and...

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Abstract

The invention provides an inactivated vaccine strain CH-GD-12-2014 against the duck adenovirus type 3. The inactivated vaccine strain CH-GD-12-2014 is preserved in China Center for Type Culture Collection, Wuhan University, Wuhan City, China, on November 27, 2017, with an accession number of CCTCC No. V201764. The whole gene sequence of the strain is submitted to Genebank, with an accession numberof KR135164. The invention also provides a polypeptide with an amino acid sequence as shown in SEQ ID No. 2, and a nucleotide sequence encoding the polypeptide, wherein the nucleotide sequence is asshown in SEQ ID No. 1. An inactivated strain provided by the invention is safe to all kinds of ducks and free of side reactions. The vaccine prepared from the inactivated strain is safe and effective,can prevent attacks caused by homologous virulent viruses, and has practical and good application value.

Description

technical field [0001] The invention relates to an inactivated vaccine, in particular to a duck adenovirus type 3 inactivated vaccine strain CH-GD-12-2014, and belongs to the field of biotechnology. Background technique [0002] Poultry adenovirus infection mainly causes chicken egg drop syndrome, chicken inclusion body hepatitis, broiler chicken and turkey respiratory syndrome, quail bronchitis, young turkey hemorrhagic enteritis, turkey viral hepatitis, pheasant marble spleen disease, etc. . The infection of avian adenoviruses has been increasing year by year, especially in the past two years, the incidence of avian adenoviruses characterized by inclusion body hepatitis has become increasingly serious. According to different group-specific antigens, avian adenoviruses are divided into groups I, II and III. Group I avian adenoviruses include traditional adenoviruses and other poultry isolates, which have a common group antigen and a total of 12 serotypes. Common ones incl...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/235A61P31/20C07K14/075C12N15/34C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552C07K14/005C12N7/00C12N2710/10221C12N2710/10222C12N2710/10234
Inventor 谢青梅张新珩周镇海李鸿鑫左珂菁
Owner SOUTH CHINA AGRI UNIV
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