New porcine reproductive and respiratory syndrome virus ORF5 modified gene and application thereof
A respiratory syndrome and ORF5 technology, applied in the field of molecular biology, can solve the problems of unsatisfactory immune protection efficiency, low expression efficiency, and reduced antibody level of GP5 gene
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0040] Example 1 Construction and Identification of Porcine Reproductive and Respiratory Syndrome Virus ORF5 Gene Modification and Its Eukaryotic Expression Plasmid
[0041] 1.1 Primer design
[0042] According to the GP5 gene sequence of PRRSV SY0608 strain (GenBank:: EU144079.1), a pair of specific primers SY GP5.1 and SY GP5.2 were designed and synthesized, and pShuttle-GP5 plasmid (Li Yufeng, Jiang Ping, et al. Pig Reproduction and Respiration Construction and immunogenicity determination of recombinant adenovirus with GP5 protein of syndrome virus [J]. Chinese Virology, 2006, 21(4): 364-367) as template to amplify GP5 gene.
[0043] Upstream primer SY GP5.15'-ATTCTCGAGATGTTGGGGAAGTGC-3' (SEQ ID NO.6),
[0044] The downstream primer SY GP5.25'-CGATTCTAGACTAGAGACGACCCCATT-3' (SEQ ID NO.7), introduces restriction enzyme cutting sites XhoI and XbaI at the 5' ends of the upstream and downstream primers respectively. The above primers were provided by Shanghai Yingjun Biotechn...
Embodiment 2
[0056] Example 2 Study on immune characteristics of eukaryotic plasmid DNA mice expressing modified GP5 protein
[0057] 2.1 Mass preparation of eukaryotic expression plasmids
[0058] A large number of plasmids were extracted by alkaline cleavage method, OD260 and OD280 were measured with a spectrophotometer, and the content and purity of each recombinant eukaryotic plasmid were calculated. DNA content (μg / mL) = sample dilution factor × 50 × OD260.
[0059] 2.2 Grouping of mice and immunization experiments
[0060] Seventy female Balb / c mice aged 6-8 weeks were divided into 7 groups with 10 mice in each group. The first group is the negative control group, injected with PBS, and the second, third, fourth, fifth, sixth, and seventh groups were injected with pCI-neo, pCI-GP5, pCI-SynGP5, pCI-SynGP5 / GP4-5, pCI-SynGP5 / GP4-5, The dose of pCI-SynGP5 / N-7 and pCI-SynGP5 / GP4-3 was 100 μg per mouse, and the same dose was boosted with the same dose on the 21st and 42nd days after the...
Embodiment 3
[0070] Example 3 Eukaryotic plasmid expressing modified GP5 protein and its recombinant attenuated Salmonella immune characteristics of piglets
[0071] 3.1 Construction and identification of recombinant attenuated Salmonella
[0072] Prepare competent cells of attenuated Salmonella SL3263 (attenuated strain of Salmonella choleraesuis C500, China Veterinary Drug Administration, the same below), and then transfer the recombinant plasmid into the attenuated Salmonella SL3263 by electroporation, after transformation, coat the transformed product LB plates containing 50 μg / mL ampicillin resistance were cultured overnight at 37°C. Pick a single transformed colony and culture it in LB liquid medium containing 50 μg / mL ampicillin until OD600nm=1.0; extract the plasmid by alkaline cleavage method, and then identify it by double enzyme digestion, which proves that the recombinant attenuated Salmonella SL-pCI-SynGP5 was obtained / GP4-5( Figure 9 ).
[0073] 3.2 Pig immune and challe...
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