Chicken IL-18 (Interleukin-18) and Newcastle disease virus HN (Hemagglutinin-neuraminidase) gene recombinant fusion protein and application
A technology of Newcastle disease virus and fusion protein, which is applied in the field of genetic engineering to produce vaccines and immune adjuvants, can solve the problems of unsatisfactory animal protection experiments, and achieve the effects of easy mass production, good protection, and mass production
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1c
[0053] Example 1 Obtaining of chIL-18-HN fusion protein coding gene sequence
[0054] 1. Acquisition of poultry IL-18 gene sequence
[0055] According to the nucleotide sequence of poultry IL-18 in GenBank, a pair of specific primers F was designed using DNA Star7.0 and Primer premier6.0 software 1 , F 2 Amplifies IL-18. Primers were synthesized by Dalian Bao Biological Engineering Company. A NotI endonuclease site was introduced at the F15' end of the primer, a BamHI endonuclease site was introduced at the F25' end and a flexible linker was simultaneously introduced. The primer sequences are as follows:
[0056] F1: SEQ ID NO.1;
[0057] P1: 5′- GCGGCCGCG ATGCCTTTTGTAAGGATA-3′
[0058] F2: SEQ ID NO.2;
[0059] P2: 5′-GGT GGATCC GGCGGTTCTGGCGGATTATAGGTTGTGCCTTTCATTATG-3′
[0060] The NDV-I vaccine was diluted 50 times, inoculated into 10-day-old chicken embryos through the allantoic cavity, 0.2mL / piece, and incubated at 37°C. Undead chicken embryos were taken out...
Embodiment 2c
[0072] Example 2 Construction of chIL-18-HN Fusion Gene Recombinant Yeast Expression Vector
[0073]Use Not I and Kpn I endonucleases to perform double enzyme digestion on the PCR product of the above chIL-18-HN fusion gene and the Pichia pastoris expression vector pPICZαA. After the digestion products are also identified by 1% agarose gel electrophoresis, the gel recovery reagent boxes for recovery identification. The digested chIL-18-HN fusion gene and Pichia pastoris expression vector pPICZαA were ligated overnight at 4°C at a molar ratio of 1:3. Take the ligation product and add it to a polypropylene centrifuge tube containing 100 μL of competent DH5α, mix gently and then ice-bath for 30 min. After the polypropylene centrifuge tube was taken out of the ice, it was heat-shocked at 42°C for 90 sec, and then immediately ice-bathed for 2 min. Add 800 μL of LB medium preheated at 37°C, and shake (100-150 rpm) at 37°C for 45 minutes. Take 100 μL of the bacterial liquid and ev...
Embodiment 3
[0074] Example 3 Construction of a genetically engineered strain expressing chIL-18-HN fusion protein:
[0075] The positive recombinant yeast expression vector pPICZαA-chIL-18-HN obtained in Example 2 was linearized with SacI, and 5 μg of the linearized recombinant expression vector pPICZαA-chIL-18-HN was mixed with 80 μL of competent Pichia pastoris X-33 , transferred to a pre-cooled 0.2cm electro-cup, placed on ice for 5min, 1.5kV, 25μF, 200Ω electric shock, immediately added 1mL of pre-cooled 1mol / L sorbitol, took 200μL and spread it on a YPDS plate, cultured at 30℃ until single Colonies appear; refer to Pichia Expression Kit for detailed steps; use PCR method to analyze Pichia transformants, prepare PCR template by boiling-freezing-boiling method, use primers F1 and F4: the reaction system is the same as above, PCR reaction conditions: 94 ° C for 5 min ; 94°C for 45s, 48°C for 45s, 72°C for 45s, 25 cycles; 72°C for 6 minutes; the clone with a size of about 2200bp was iden...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - 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.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap