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Novel genetic engineering vaccine for poultry egg drop syndrome virus, its preparation method and application

A technology for egg drop syndrome and viruses, applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of poor protein folding, mixing, and poor immunogenicity, and achieve high immunogenicity, Low production cost and strong immunogenic effect

Active Publication Date: 2022-06-24
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN108653724A discloses a kind of preparation method of the fiber protein gene engineering subunit vaccine of avian egg drop syndrome virus expressed with Escherichia coli expression system, and CN106232813A discloses a kind of subunit vaccine expressed fusion protein with E. The protein expressed by the expression system has poor foldability and poor immunogenicity, and E. coli itself contains endotoxin and toxic proteins, which may be mixed in the final product

Method used

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  • Novel genetic engineering vaccine for poultry egg drop syndrome virus, its preparation method and application
  • Novel genetic engineering vaccine for poultry egg drop syndrome virus, its preparation method and application
  • Novel genetic engineering vaccine for poultry egg drop syndrome virus, its preparation method and application

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preparation example Construction

[0060] One aspect of the embodiments of the present invention provides a method for preparing an immune composition, comprising:

[0061] The coding genes of the recombinant avian egg reduction syndrome virus Core protein and Fiber protein are cloned into the shuttle vector to obtain the first recombinant shuttle vector;

[0062] The coding genes of the recombinant avian egg reduction syndrome virus Penton protein and Hexon protein are cloned into the shuttle vector to obtain the second recombinant shuttle vector;

[0063] The first recombinant shuttle vector and the second recombinant shuttle vector are respectively transformed into DH10Bac bacteria to obtain the first recombinant baculovirus vector and the second recombinant baculovirus vector;

[0064] Using the first recombinant baculovirus vector and the second recombinant baculovirus vector to transfect insect cells respectively to obtain a recombinant baculovirus;

[0065] Insect cells were co-infected with the recombi...

Embodiment 1

[0123] Example 1 Construction and identification of transfer vector Dual-C-F

[0124] 1. Construction and identification of transfer vector Dual-C

[0125] 1.1 C gene amplification and purification The codon-optimized C gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Company and cloned into the pUC17 vector to obtain the pUC-C plasmid vector. The pUC-C plasmid was used as a template, and C-F and C-R were used as upstream and downstream primers for PCR amplification (the gene sequences of C-F and C-R are shown in SEQ ID NOs: 9 and 10). The amplification system is shown in Table 1.

[0126] Table 1 C gene amplification system

[0127]

[0128] The reaction conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 45 seconds, annealing at 54°C for 45 seconds, extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 10 minutes.

[0129] The PCR product is subjected to gel electrophoresis to verify the size of the target gene, such as ...

Embodiment 2

[0159] Example 2 Construction and identification of transfer vector Dual-P-H

[0160] 1. Construction and identification of transfer vector Dual-P

[0161] 1.1 P gene amplification and purification The codon-optimized P gene (SEQ ID NO: 5) was synthesized in Nanjing GenScript Company and cloned into the pUC17 vector to obtain the pUC-P plasmid vector. The pUC-P plasmid was used as a template, and P-F and P-R were used as upstream and downstream primers for PCR amplification (the gene sequences of P-F and P-R are shown in SEQ ID NOs: 13 and 14). The amplification system is shown in Table 9.

[0162] Table 9 P gene amplification system

[0163]

[0164] The reaction conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 45 seconds, annealing at 54°C for 45 seconds, extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 10 minutes.

[0165] The PCR product is subjected to gel electrophoresis to verify the size of the target gene, such as ...

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Abstract

The invention discloses a preparation method of an antigenic protein, comprising: respectively cloning the coding genes of codon-optimized recombinant avian egg drop syndrome virus Core protein, Fiber protein, Penton protein and Hexon protein into multiple recombinant shuttle vectors; Respectively transform the multiple recombinant shuttle vectors into competent host cells to obtain multiple recombinant baculovirus vectors; respectively transfect insect cells with the multiple recombinant baculovirus vectors to obtain recombinant baculoviruses; Co-infect insect cells to automatically assemble into VLPs in the cells and express antigenic proteins. The invention also discloses an immune composition containing the antigenic protein, which can be used to prepare a genetically engineered subunit vaccine of the avian egg drop syndrome virus. The structure, immunogenicity and function of the vaccine are similar to those of the natural protein, and the expression level is High, strong immunogenicity, no pathogenicity to animals, and can be prepared by large-scale serum-free suspension culture in a bioreactor, and the production cost is low.

Description

technical field [0001] The invention relates to a genetically engineered vaccine, in particular to a novel genetically engineered vaccine for avian egg reduction syndrome virus, a preparation method and application thereof, and belongs to the technical field of animal immunization drugs. Background technique [0002] Egg Drop Syndrome (EDS) is a viral infectious disease that reduces the egg production rate of laying hens or breeding hens. EDS has no clinical symptoms, or mild diarrhea can be seen. It mostly occurs in the 30-40 weeks of age during the peak spawning period. It will cause the infected flocks to lay eggs with deformed eggshells or decrease egg production for 3-8 weeks. Huge loss. Immunization is the main measure to prevent this disease. All the chickens will produce antibodies before entering the laying eggs, and the laying eggs will not be affected. [0003] Egg Drop Syndrome Virus (EDSV) belongs to avian adenovirus group III, with a single serotype and almos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/866C07K14/075C12N15/34G01N33/569A61K39/235A61P31/20
CPCC12N15/86C07K14/005G01N33/56983A61K39/12A61P31/20C12N2710/14043C12N2710/10222C12N2710/10123G01N2333/075C12N2710/10234A61K2039/5256A61K2039/552
Inventor 曹文龙孔迪滕小锘易小萍张大鹤
Owner 苏州沃美生物有限公司
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