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
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[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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