Preparation and application of baculovirus expression system-based duck tembusu virus subunit vaccine
A technology of baculovirus and recombinant baculovirus, applied in the direction of microorganism-based methods, applications, vaccines, etc., can solve the problems of immune effect being easily affected by various factors, poor protein biological activity, strong virulence, etc. , to achieve good protective effect, high biological safety and good immunogenicity
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[0078] Example 1: Construction of recombinant baculovirus AC-prME expressing duck tambusu PrM-E gene
[0079] 1. Construction of recombinant baculovirus AC-prME
[0080] 1. Acquisition of prME gene
[0081] According to the prME gene and signal peptide sequence of Duck Tembusu virus DF2 strain (Duck Tembusu virus DF2), a 2100bp prME gene and signal peptide sequence (GenBank ID: KJ489355) were artificially synthesized, and the sequence was cloned into the vector pUC-18 (Invitrogen) Obtain the recombinant plasmid pUC-prME. Use this plasmid as a template with primer pairs
[0082] Forward: 5’-TAGGCGGCCGCATGCAGATGCTCGACGGACTGAAT-3’,
[0083] Reverse: 5’-AGCTGCAGTTAGGCATTGACATTTACTGC-3’;
[0084] Carry out PCR amplification and recover the prME gene fragment with a size of 2100bp.
[0085] 2. Construction of baculovirus transfer vector pFastBac1-prME
[0086] Baculovirus universal vector pFastBac1 TM (Invitrogen) as the backbone, using NotI+PstI (purchased from Bao Biology (Dalian) Co., Ltd.)...
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[0118] Example 2: Subunit vaccine prepared by recombinant baculovirus Ac-prME
[0119] 1. Subunit vaccine prepared by recombinant baculovirus Ac-prME.
[0120] The recombinant baculovirus Ac-prME obtained in Example 1 was used to inoculate suspension cultured insect cells sf9 at a multiplicity of infection of 2.0 MOI. After 72 hours, the cells and supernatant were collected, and the cells were purified as described in Example 1 in 4 After measuring the protein concentration, the water phase is composed of protein and Tween 80 in a volume ratio of 97:3, and the water phase and oil phase are mixed and emulsified at a volume ratio of 1:2 to prepare a sub-vaccine. The total protein content is 0.5 mg / ml, which is used in Example 3 and Example 4 below, and stored at 4 degrees.
[0121] 2. Subunit vaccine prepared by recombinant baculovirus Ac-prME.
Example Embodiment
[0123] Example 3: Subunit vaccine composition and routine inspection
[0124] Dosage form: water-in-oil type (W / O)
[0125] Adjuvant composition: white mineral oil (Marcol52)
[0126] Tween 80 (CRILLET4)
[0127] Vaccine emulsification: The water phase is composed of protein and Tween 80 in a volume ratio of 97:3, and the water phase and oil phase are mixed and emulsified in a volume ratio of 1:2 to prepare a sub-vaccine.
[0128] Vaccine character detection: After emulsification, the vaccine is dropped into clean water, the first drop is dispersed, and the second drop is oily. The oily drops will not break when shaking the liquid surface.
[0129] Sterility test: Take a small amount of vaccine to coat TSA plate, culture in 37℃ incubator for 18 hours, aseptic colony grows.
[0130] Stability test: Centrifuge at 3000rpm / min for 15 minutes. After centrifugation, the emulsified vaccine is not stratified.
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