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Novel genetic engineering vaccine for avian egg drop syndrome virus as well as preparation method and application of novel genetic engineering vaccine

A technology for egg reduction syndrome and virus, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of poor protein folding, poor immunogenicity, mixed and other problems

Active Publication Date: 2021-04-27
苏州世诺生物技术有限公司
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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 avian egg drop syndrome virus as well as preparation method and application of novel genetic engineering vaccine
  • Novel genetic engineering vaccine for avian egg drop syndrome virus as well as preparation method and application of novel genetic engineering vaccine
  • Novel genetic engineering vaccine for avian egg drop syndrome virus as well as preparation method and application of novel genetic engineering vaccine

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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, which includes:

[0061] Cloning the coding genes of recombinant avian egg drop syndrome virus Core protein and Fiber protein into the shuttle vector to obtain the first recombinant shuttle vector;

[0062] Cloning the genes encoding the Penton protein and the Hexon protein of the recombinant avian egg drop syndrome virus into the shuttle vector to obtain the second recombinant shuttle vector;

[0063] respectively transforming the first recombinant shuttle vector and the second recombinant shuttle vector 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 recombinant baculovirus;

[0065] Simultaneously inoculate insect cells with the recombinan...

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 Amplification and purification of the C gene The codon-optimized C gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript 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 NO:9 and 10). The amplification system is shown in Table 1.

[0126] Table 1 C gene amplification system

[0127]

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

[0129] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as figure 1 As shown, the ...

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 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 NO: 13 and 14). The amplification system is shown in Table 9.

[0162] Table 9 P gene amplification system

[0163]

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

[0165] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as Figure 6 As shown, the targ...

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Abstract

The invention discloses a preparation method of antigen protein. The preparation method comprises the following steps of cloning encoding genes of codon-optimized recombinant avian egg drop syndrome virus Core protein, Fiber protein, Penton protein and Hexon protein onto a plurality of recombinant shuttle vectors; transforming competent host cells by the plurality of recombinant shuttle vectors to obtain a plurality of recombinant baculovirus vectors; transfecting insect cells with the plurality of recombinant baculovirus vectors to obtain recombinant baculoviruses; and co-infecting insect cells with the recombinant baculoviruses, so that the recombinant baculoviruses are automatically assembled into VLP in the cells, and the antigen protein is expressed. The invention also discloses an immune composition containing the antigen protein. The immune composition can be used for preparing an avian egg drop syndrome virus genetic engineering subunit vaccine; and the vaccine is similar to natural protein in structure, immunogenicity and function, high in expression level and strong in immunogenicity, has no pathogenicity to animals, can be subjected to large-scale serum-free suspension culture preparation through a bioreactor, and is low in production cost.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a novel genetic engineering vaccine of poultry egg drop syndrome virus, and its preparation method and application belong to the technical field of animal immune 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 at the age of 30-40 weeks during the peak egg-laying period, and it will cause infected chickens to produce eggs with deformed eggshells or a decrease in egg production for 3-8 weeks. Therefore, economical The loss is huge. Immunization is the main measure to prevent this disease. All chickens will produce antibodies before laying eggs, and laying eggs will not be affected. [0003] Egg Drop Syndrome Virus (EDSV) belongs to avian adenovirus group III, with a single serotype and almost n...

Claims

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Application Information

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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 苏州世诺生物技术有限公司
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