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Vaccine of swine fever-porcine contagious pleuropneumoniae bivalent subunit and preparation method and applications thereof

A technology of porcine pleuropneumonia and pleuropneumonia, which is applied in the field of vaccine and its preparation of two subunits of swine fever-porcine infectious pleuropneumonia, and can solve the problem of high cost of immunization alone

Active Publication Date: 2019-07-23
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Both swine fever and porcine contagious pleuropneumonia have high cost of immunization alone, and there is currently no effective measure to immunize both diseases at the same time

Method used

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  • Vaccine of swine fever-porcine contagious pleuropneumoniae bivalent subunit and preparation method and applications thereof
  • Vaccine of swine fever-porcine contagious pleuropneumoniae bivalent subunit and preparation method and applications thereof
  • Vaccine of swine fever-porcine contagious pleuropneumoniae bivalent subunit and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Synthesis, PCR amplification and identification of embodiment 1 classical swine fever virus E2 gene

[0079]Downloaded the published E2 sequence of classical swine fever virus from GenBank and the E2 sequence of new strains popular in recent years as a reference, carried out codon optimization and modification on the basis of the E2 gene of the C strain, and added a signal peptide sequence (VLRGQIVQGVIWLLLVTGAQG , SEQ ID NO.11), the 3' end is connected with a His tag composed of 6 histidines, and the modified sequence is synthesized by a gene synthesis company; after obtaining the synthesized E2 gene through PCR amplification, the amplified The product E2 gene was sequenced, and the measured E2 gene was compared with the E2 gene of the C strain and the reference strains of each subgroup. The results showed that the nucleotide homology between the finally obtained E2 gene and the E2 gene of the C strain Reaching 93.2%, as the target gene for constructing CSFV E2 recombin...

Embodiment 2

[0085] Example 2 Construction and identification of recombinant baculovirus of classical swine fever virus E2 protein

[0086] The construction method of the recombinant baculovirus of classical swine fever virus E2 protein is shown as a flow chart figure 2 shown. Using the synthetic swine fever virus E2 gene as a template, high-fidelity Pfu DNA polymerase was used for PCR reaction to amplify the swine fever virus E2 gene. After sequencing and verification, the recombinant virus was constructed by referring to the Invitrogen operation manual. Gene amplification and identification, construction of transfer vector, screening and identification of transfer vector, transfection of transfer vector and linear baculovirus, screening of recombinant virus, identification of recombinant virus and expression of E2 protein, and finally obtain a virulence strain The stable recombinant baculovirus with high E2 protein secretion was named CSFV-Rb-E2.

[0087] 1) Amplification of E2 gene ...

Embodiment 3

[0103] Expression condition optimization and harvest of embodiment 3 classical swine fever virus E2 protein

[0104] Using a 3×3 orthogonal experiment design, the three factors of inoculation dose, cell density at inoculation, and harvest time were compared and analyzed, among which three levels of inoculation doses were set at 0.1, 0.5, and 1 MOI, and 1 million, 2 million, and 3 million / There are three levels of cell density in ml, and the three conditions of collection time are 72, 96, and 120 hours. The E2 protein content in the supernatant harvested from the above experiments is detected by the double-antibody sandwich ELISA method. Finally, it was determined that when the Sf-9 cell density was 2 million / ml, it was inoculated with 1 MOI, and the expressed recombinant E2 protein had the highest content. It was verified by Western-blot that the protein had good immunogenicity. SDS-PAGE protein electrophoresis and gelation The gel imaging system comes with band analysis soft...

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Abstract

The invention relates to the technical field of veterinary drugs, in particular to a vaccine of swine fever-porcine contagious pleuropneumoniae bivalent subunit and a preparation method and applications thereof. The vaccine comprises classical swine fever virus E2 protein, serum type-7 actinobacillus pleuropneumoniae toxin protein ApxII, serum type-1 actinobacillus pleuropneumoniae outer membraneprotein Oml and vaccine adjuvant. The vaccine is obtained by mixing and emulsifying the proteins and the vaccine adjuvant in equal volume. The vaccine can simultaneously prevent swine fever and porcine contagious pleuropneumoniae caused by serum type-1 and type-7 porcine actinobacillus pleuropneumoniae, and has good effect and low cost.

Description

technical field [0001] The invention relates to the technical field of veterinary medicine, in particular to a vaccine of a dual subunit of swine fever-porcine infectious pleuropneumonia, a preparation method and application thereof. Background technique [0002] Classical swine fever is a highly contagious severe contagious disease of swine fever caused by swine fever virus, which causes huge economic losses to the swine industry every year. Although the current attenuated CSF vaccine has controlled the large-scale epidemic of CSF, CSF is still spreading sporadically. Due to the lack of effective vaccine immunization and differential diagnosis methods for epidemic strain infection, it is necessary to develop and promote the use of labeled vaccines such as E2 subunit vaccines for the purification of classical swine fever. [0003] Different from traditional attenuated vaccines, the main antigen of CSF E2 vaccine is CSFV E2 protein. E2 protein is one of the structural prote...

Claims

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Application Information

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IPC IPC(8): A61K39/187A61K39/102A61K39/39A61P31/04A61P31/14
CPCA61K39/12A61K39/102A61K39/39A61P31/04A61P31/14A61K2039/70A61K2039/552C12N2770/24334A61K2300/00Y02A50/30
Inventor 贺笋王遵宝郑培李俊辉豆智华曹剑张飞唐慧芬师小潇
Owner 天康生物制药有限公司
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