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Duck short beak dwarf syndrome gene engineering subunit inactivated vaccine and preparation method thereof

An inactivated vaccine, genetic engineering technology, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of high biological safety risk, high side effects, high labor intensity, etc.

Pending Publication Date: 2021-06-11
北京维牧康动保生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The incidence of the disease ranges from 10% to 100%, and the mortality rate is lower than 10%, but the rate of stiff ducks or dead ducks is as high as 20% to 80%. The prevalence of the disease has caused huge economic losses to the duck industry in my country.
[0004] Due to the mutation of the virus, the currently used gosling plague vaccine or antibody products cannot effectively prevent and control the prevalence of the disease in ducks, so it is urgent to develop a vaccine against the mutant strain
The invention isolates the mutated virus strain, optimizes the codon of its VP2 gene, successfully constructs the recombinant baculovirus, and prepares the genetically engineered subunit inactivated vaccine for duck short-beaked dwarf syndrome, aiming at immunizing ducklings so that ducklings can obtain Good passive immunization not only fills the gap in the prevention and control of mutant strains, but also solves the problems of low protection rate, high labor intensity, high cost, high biosafety risk and the difficulty of virus cultivation in whole virus inactivated vaccines existing in antibody products , high side effects and other issues

Method used

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  • Duck short beak dwarf syndrome gene engineering subunit inactivated vaccine and preparation method thereof
  • Duck short beak dwarf syndrome gene engineering subunit inactivated vaccine and preparation method thereof
  • Duck short beak dwarf syndrome gene engineering subunit inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1—Isolation and identification of duck short beak type gosling plague virus SBDS-GPV JS01 strain

[0053] In April 2018, the company isolated a strain of duck short-beaked gosling plague virus from a duck farm in Jiangsu, which was named SBDS-GPV JS01 strain. The virus strain can be continuously passed down in duck embryos, and when passing on to the 5th generation (P5), the duck embryos died 100% within 7 days after receiving the virus, and the dead embryo body lesions were growth retardation and hemorrhage ( figure 1 ). Virus titer up to 5.3log 10 ELD 50 / mL.

[0054] The homology analysis of the VP1 gene sequence of the strain shows that the VP1 gene of the strain is identical to the newly popular short-beaked gosling plague virus strains in China (SBDS-GPV M15 strain, SBDSV-SDLC01 strain and SBDSV-DS15 strain, etc.) The homology is more than 96%, and the homology with SBDS-GPV M15 strain is the highest, reaching 99.6%. The results of the phylogenetic tr...

Embodiment 2

[0055] Example 2——Establishment of the SBDS-GPV JS01 strain to challenge the virus model of 2-day-old test ducks

[0056] Forty-six 2-day-old GPV-negative healthy Cherry Valley ducks (Table 1) were divided into 2 groups. The first group is the challenge group, containing 13 male ducks and 11 female ducks, and each duck is inoculated with 0.2mL P8 generation JS01 strain virus liquid (5.7log 10 ELD 50 / mL), 5.0log 10 ELD 50 / only. The second group is the negative control group, including 12 male ducks and 10 female ducks, and each duck is inoculated with an equal volume of sterilized PBS in the same way. Each group was kept in isolation and fed ad libitum. After the challenge, the clinical symptoms of the test ducks were observed every day for 21 consecutive days. The beak length and body weight of each duck were measured on the 7th, 14th and 21st days after challenge. At 21dpc, all test ducks were culled, dissected and further confirmed for sex.

[0057] Three days aft...

Embodiment 3

[0066] Example 3——Construction and Identification of Recombinant Baculovirus rBac-JS01 VP2 Strain

[0067] The inventors optimized the codons of the VP2 gene of SBDS-GPV JS01 strain, cloned the optimized VP2 gene (SEQ ID No.1) into a baculovirus vector, successfully constructed a recombinant baculovirus and named it duck-short-beaked gosling Pestivirus recombinant rBac-JS01 VP2 strain. By indirect immunofluorescence analysis ( Figure 4 ) and western blot analysis ( Figure 5 ), confirming that rBac-JS01 VP2 can successfully express VP2 protein in Sf9 or Sf+ cells. Electron microscope observation results ( Image 6 ) showed that the expressed VP2 protein could self-assemble into virus-like particles (VLPs).

[0068] SEQ ID No.1

[0069]

[0070]

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Abstract

The invention relates to a duck short beak dwarf syndrome gene engineering subunit inactivated vaccine and a preparation method thereof. Aiming at a variant strain, the VP2 gene of the SBDS-GPV JS01 strain is subjected to codon optimization and is cloned to a baculovirus vector, so that a recombinant baculovirus rBac-JS01VP2 strain is successfully constructed; insect cell full suspension (or spinner bottle) culture is adopted, the technology is mature, and production transformation and step-by-step amplification culture are facilitated; compared with traditional duck embryo or duck embryo fibroblast culture, the virus titer is high, the quality is stable, the production period is short, and the cost is low; compared with an antibody product, the protection rate is high, the duration time of a maternal antibody is long, only female ducks are immunized, and labor and cost are saved; serum or egg source miscellaneous protein does not exist, and the side reaction is extremely low; the expressed VP2 protein can be assembled into virus-like particles (VLPs), and the immunogenicity is good; the expression quantity is high (when the virus titer is 8.0 Log10TCID50 / ml, the VP2 protein expression quantity reaches 50-100 mg / L), the antibody level is high, and the lasting time is long; the shelf life of the product is 18 months, and the immune period is 6 months; and the ELISA antibody detection method is used for screening susceptible ducks and evaluating efficacy.

Description

technical field [0001] The invention relates to a genetic engineering subunit inactivated vaccine for duck short beak dwarf syndrome and a preparation method thereof, belonging to the field of veterinary biological products. Background technique [0002] Goose parvovirus (GPV), also known as Derzsy's disease, commonly known as goose plague, goose enteritis, etc., is a highly contagious disease that affects young geese and Muscovy ducks. The diversity of disease names reflects the The multiple pathological features of the disease is a viral infectious disease that harms many species of waterfowl. As early as 1956, Mr. Fang Dingyi was the first to discover and identify GPV in a flock of infected geese in Yangzhou City, Jiangsu Province. Afterwards, the disease spread around the world, but Muscovy ducks rarely developed the disease. [0003] Duck short beak dwarf syndrome virus (SBDSV) is also known as novel goose parvovirus (NGPV) or short beak type goose parvovirus variants....

Claims

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Application Information

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IPC IPC(8): A61K39/23A61K39/39A61P31/20C12N15/866C12N15/35
CPCA61K39/12A61K39/39A61P31/20C12N15/86C07K14/005A61K2039/5252A61K2039/552C12N2710/14143C12N2800/105C12N2750/14334C12N2750/14322
Inventor 田红荣先锋孙雅鑫
Owner 北京维牧康动保生物科技有限公司
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