rBCG for expression of Br. Melitensis P39 and L7/L12 fusion gene and construction method thereof
A P39-L7 and fusion gene technology, applied in the field of genetic engineering and vaccine preparation, to achieve significant immune adjuvant effect, save costs, and solve the effect of poor immune effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0032] Embodiment 1 carries the construction of the recombinant expression vector of Brucella melii P39 and L7 / L12 fusion gene
[0033] Include the following steps:
[0034] 1. According to the published sequences of genes P39 and L7 / L12 of Brucella melis M5 strain (GenBank: EF189139.1; GenBank: EF173477.1), after codon optimization using Jcat software, artificially synthesized full sequence optimization The latter P39-L7 / L12 fusion gene was used as the target gene. (The nucleotide sequence of the fusion gene is shown in SEQ ID NO:1).
[0035] 2. Construction of a recombinant expression vector carrying the optimized M5 strain P39-L7 / L12 fusion gene: the above target gene was inserted into the shuttle expression vector pMV361 through two restriction sites of PvuII and BstBI.
[0036] 3. Verification of inserting the correct recombinant expression vector.
[0037] Through nucleic acid sequence determination, it was confirmed that the recombinant expression vector carrying the...
Embodiment 2
[0038] Example 2 Construction of rBCG expressing Brucella melis P39 and L7 / L12 fusion gene
[0039] 1. Using BCG as the host bacterium, transform the recombinant expression vector constructed in Example 1 (full sequence shown in SEQ ID NO: 2) into BCG.
[0040] The electroporation method was used for transformation, and the experimental conditions were: 2500V, 25μF, 1000Ω, electroporation time 5ms, 0.1cm electric shock cup. The electroporation reaction system is: 3 μl of plasmid (concentration is 0.68 μg / μl), 100 μl of competent BCG bacterial solution (concentration is about 1×10 10 CFU / ml).
[0041] 2. Screening of positive clones
[0042] After electroporation, they were inoculated on a medium (slant) containing 50 μg / ml kanamycin for positive clone selection.
[0043] 3. Detection of target gene expression
[0044]The screened positive clones (ie, recombinant BCG) were inoculated into liquid medium for expansion culture, the culture supernatant was collected, and the ex...
Embodiment 3
[0045] Effect experiment of embodiment 3 brucellosis vaccines
[0046] Immunize female Balb / c mice aged 6-8 weeks with recombinant BCG and inject subcutaneously at a dose of 4×10 8 CFU / mouse, 4 weeks after immunization, the expression of Th1 / Th2 cytokines in the serum of mice in each group was detected. The experimental results showed that, compared with recombinant BCG carrying unoptimized P39-L7 / L12 fusion gene (rBCG-P39-L7 / L12(wild)) and untransformed BCG, carrying codon-optimized P39-L7 / L12 The recombinant BCG with fusion gene (rBCG-P39-L7 / L12) can effectively induce the production of Th1 cytokines such as IL-2, IL-12 and IFN-γ. ( figure 2 )
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com