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
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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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