Chicken infectious anemia subunit vaccine

A chicken infectious anemia, subunit vaccine technology, applied in vaccines, veterinary vaccines, single-stranded DNA viruses, etc. The effect of preventing infection and improving the level of immune antibodies

Inactive Publication Date: 2019-07-19
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because chicken infectious anemia virus can cause immunosuppression, the immune effect of inactivated vaccine is not ideal
However, the attenuated live vaccine has residual virulence and the possibility of strong virulence, and its safety is questioned

Method used

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  • Chicken infectious anemia subunit vaccine
  • Chicken infectious anemia subunit vaccine
  • Chicken infectious anemia subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, the screening of chicken infectious anemia VP1, VP2 protein

[0018] In 2016, typical chicken infectious anemia symptoms appeared in a chicken farm in Shandong Province. In order to isolate the pathogen, aseptically collect the thymus of the diseased chicken, homogenize it with sterile saline to make a suspension, centrifuge to collect the cleared bacteria, and then inoculate 6.5-day-old SPF embryos through the allantoic cavity, hatch for 264 hours, and collect the embryo body tissue , after homogenization, repeated freezing and thawing, take the supernatant and freeze it. After the harvested virus liquid was purified, the analysis and detection of virus characteristics in terms of virus content, immunogenicity, specificity and purity were carried out. The results showed that the isolated virus strain had a specific reaction with chicken infectious anemia and had no bacteria or mycoplasma. and exogenous virus contamination.

[0019] The character of the ...

Embodiment 2

[0023] Embodiment 2, the construction of the recombinant baculovirus expressing VP1, VP2 gene

[0024] 2.1 Cutting and optimization of VP1 and VP2 genes

[0025] Cut off 32aa at the N-terminus of the VP1 gene, cut off 7aa at the C-terminus, and mutate the 173rd M to P, and the 304th W to S in the conserved sequence, so as to help the protein to better fold into a tertiary structure, To better expose its antigenic site, add a start codon to the N-terminal; the sequence of the amino acid after optimization and modification is SEQ ID NO: 5, which can well express the antigenic site; at the same time, the codon Optimized for better expression; the nucleotide sequence of the optimized VP1 gene is SEQ ID NO:6. The VP2 gene was codon-optimized, and the nucleotide sequence of the optimized gene is SEQ ID NO:7.

[0026] 2.2 Construction of VP1 positive plasmid

[0027] 2.2.1 Enzyme digestion reaction

[0028] 2.2.1.1 Mark the 1.5mL EP tube to be used, and add and mix the sample acc...

Embodiment 3

[0097] Embodiment 3: the packing of baculovirus

[0098] (1) Preparation: UV sterilization in a biosafety cabinet for 30 minutes; TNM-FH culture solution was placed in a 27°C water bath and preheated to 27°C.

[0099] (2) Add 2 μg of recombinant DNA to 100 μl of TNM-FH medium without serum and double antibody, and mix well. Add 9 μl Cellfectin Reagent to 100 μl TNM-FH medium without serum and double antibody, and mix well. The liposomes were mixed with the recombinant DNA and allowed to stand at room temperature for 40 min.

[0100] (3) Take out the 6-well plate cells from the incubator at 27°C, discard the supernatant medium, wash the cells three times with pre-warmed TNM-FH culture medium, and discard the TNM-FH culture medium.

[0101] (4) Add 2 ml of 10% fetal bovine serum TNM-FH culture solution to each cell well.

[0102] (5) Gently add the mixture of recombinant DNA and liposomes into each well of cells, mix gently, and culture statically at 27°C for 5-6h.

[0103] ...

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Abstract

The invention provides VP1 protein and VP2 protein for chicken infectious anemia viruses, wherein the amino acid sequence of the VP1 protein is SEQID NO:5, and the amino acid sequence of the VP2 protein is SEQID NO:4. In another respect, the invention provides recombinant baculovirus which carries a nucleotide fragment coding the VP1 protein and the VP2 protein. The constructed recombinant baculovirus is used for expressing the VP1 protein and the VP2 protein for the chicken infectious anemia viruses in insect cells. The invention further provides a chicken infectious anemia virus subunit vaccine. The chicken infectious anemia virus subunit vaccine includes an antigen and a vaccine adjuvant, wherein the used antigen is the VP1 protein and the VP2 protein prepared in the invention. After the prepared vaccine is used for immunizing laying hens, the level of immune antibodies can be increased, the maternal antibody level of filial generations is guaranteed, and infection with the chickeninfectious anemia viruses can be prevented.

Description

technical field [0001] The invention belongs to the technical field of poultry vaccine preparation, in particular to a chicken infectious anemia virus subunit vaccine. Background technique [0002] Chicken infectious anemia virus is a common immunosuppressive virus that can cause thymus atrophy, aplastic anemia, muscle and subcutaneous tissue hemorrhage in chicks, and chickens of all ages are susceptible to infection. Avian infectious anemia is a major global infectious disease that causes immunosuppression, high mortality from secondary infections, and makes other vaccines less effective. The disease has broken out in South Korea, Argentina, Nigeria, Hungary and other countries. The positive rate of chicken farms in Guangdong, Beijing, Zhejiang, Shanghai and other regions in China has also reached 42%. Therefore, it is urgent to develop corresponding vaccines to prevent and control the disease. [0003] Chicken infectious anemia virus belongs to the family Circoviridae, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C12N7/01A61K39/12A61P31/20
CPCA61K39/12A61K2039/552A61P31/20C07K7/00C07K14/005C12N2710/14021C12N2750/10022C12N2750/10034
Inventor 郭伟伟向银辉刘大卫陈俭梅赵鹏韩乃君范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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