Subunit vaccine of PPV (porcine parvovirus) disease and preparation method of subunit vaccine

A parvovirus and vaccine technology, applied in the field of microorganisms and veterinary biopharmaceuticals, can solve problems such as unstable immune effects and biosafety risks, and achieve good immune and preventive effects, high immunogenicity, and enhanced immune effects

Inactive Publication Date: 2018-04-03
CHANGCHUN SR BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inactivated vaccine has unstable immune effect, and the whole vaccine preparation process needs to deal with a large number of infectious viruses, so there is a certain biosafety risk, so it is not the most ideal vaccine.

Method used

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  • Subunit vaccine of PPV (porcine parvovirus) disease and preparation method of subunit vaccine
  • Subunit vaccine of PPV (porcine parvovirus) disease and preparation method of subunit vaccine
  • Subunit vaccine of PPV (porcine parvovirus) disease and preparation method of subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation and identification of porcine parvovirus virus-like particles

[0025] The preserved porcine parvovirus genome was sequenced to obtain the VP2 sequence, optimized according to the preference of Sf9 cells, and sent to the company for gene synthesis. The optimized porcine parvovirus VP2 sequence is shown in SEQ ID NO.3.

[0026] For the optimized sequence, use primers (shown in SEQ ID NO.4-5) to amplify the VP2 gene, connect it into the pH MCS of the pFastBac Dual vector through PstI-HindIII, and then introduce IL- 15 gene sequences, the nucleotide sequence of which is shown in SEQ ID NO.6. The IL-VP2 gene was amplified by PCR (primers are shown in SEQ ID NO.7-8), ligated into p10MCS using SmaI-XhoI, and finally obtained a transfer vector carrying double-copy foreign fragments. The transfer vector was transformed into DH10Bac competent for recombination, and the recombinant baculovirus genome Bacmid carrying double-copy exogenous fragments was obta...

Embodiment 2

[0028] Embodiment 2 Guinea pig immunity test

[0029] The porcine parvovirus virus-like particles prepared in Example 1 were diluted with 20mM PBS (pH 7.2) so that their hemagglutination titers were 1:2 8 and 1:2 10 , adding thimerosal at a final concentration of 0.005%, adding ISA201VG adjuvant at an equal volume ratio, emulsifying to prepare high-dose vaccine and low-dose vaccine, and storing at 4°C.

[0030] 200-300g guinea pigs were divided into 4 groups, respectively negative control group, high-dose vaccine group, low-dose vaccine group and inactivated vaccine (purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) control group, 3 / group, immunized The dose is 0.5ml / bird, and the immunization is boosted once in the 4th week after the first immunization. Blood was collected at 0, 2, 3, 4, and 6 weeks after the first immunization, and the serum hemagglutination inhibitory antibody titer was determined (for the operation method, refer to "Veterinary Biological Pr...

Embodiment 3

[0034] Embodiment 3 Pig immune test

[0035] Dilute the produced porcine parvovirus virus-like particles with 20mM PBS (pH 7.2) to make the hemagglutination titer 1:2 10 , adding thimerosal at a final concentration of 0.005%, adding ISA201VG adjuvant at an equal volume ratio, emulsifying to make a vaccine, and storing at 4°C.

[0036] Ten healthy susceptible piglets aged 4-6 weeks were randomly divided into two groups, immunization group and control group, 5 piglets per group. Piglets in the immune group were intramuscularly injected with 2 ml of the porcine parvovirus virus-like particle vaccine above. At 0, 3, and 4 weeks after immunization, blood was collected from the experimental pigs, the serum was separated, and the serum anti-porcine parvovirus hemagglutination inhibition antibody titer was determined by the hemagglutination inhibition test.

[0037] The results showed (Table 2), the VLPs vaccine immunization group could stimulate the body to produce anti-porcine par...

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Abstract

The invention provides a subunit vaccine of PPV (porcine parvovirus) disease and a preparation method of the subunit vaccine. PPV virus-like particles are provided firstly and produced by steps as follows: optimized VP2 gene and IL-15 (interleukin-15) gene are subjected to fusion expression, and the PPV virus-like particles are produced by a baculovirus/insect cell expression system. The PPV virus-like particles are mixed with a preservative and an adjuvant, and the PPV disease subunit vaccine is prepared. The PPV disease vaccine has the advantages of high yield, low production cost, high immunogenicity, good safety and the like, facilitates large-scale production, and has good immunization and prevention effects on PPV disease.

Description

technical field [0001] The invention relates to the field of microorganisms, genetic engineering and veterinary biopharmaceuticals, in particular to porcine parvovirus virus-like particles, its preparation method and application. Background technique [0002] Porcine parvovirus disease is a porcine reproductive disorder caused by porcine parvovirus (PPV) infection, which is characterized by infecting sows, especially primiparous sows, causing sows to produce stillbirths, deformed fetuses, and mummified fetuses , abortion and sickly piglets, while the sow itself has no obvious clinical symptoms. The disease was first reported in the UK in 1967 and the virus was isolated. Later, the disease was reported in many countries in Europe, America, Asia and the Atlantic Ocean. Now it has spread all over the world. In my country, Shanghai, Sichuan, Beijing and Jiangsu etc. Porcine parvovirus has also been isolated in succession. [0003] PPV is widely prevalent in pig farms in my coun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/015C12N15/62C12N15/866A61K39/23A61K39/39A61P31/20
CPCA61K39/12A61K39/39A61K2039/53A61K2039/552A61K2039/55527A61K2039/55566C07K14/005C07K2317/90C12N15/86C12N2750/14323C12N2750/14334C12N2800/105C12N2800/22
Inventor 夏振强金宏丽石晶殷玉和刘冰刘伟崔玉梅付玉
Owner CHANGCHUN SR BIOLOGICAL TECH
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