Poultry IL-2 and newcastle disease virus HN gene recombination fusion protein and application thereof
A Newcastle disease virus and fusion protein technology, which is applied in the recombinant fusion protein of poultry IL-2 and Newcastle disease virus HN gene and its application field, can solve the problems of unsatisfactory animal protection experiments and other problems
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Embodiment 1
[0068] get code chIL-2-HN Gene sequence of fusion protein:
[0069] According to the nucleotide sequence of avian IL-2 in GenBank (accession number: AY029588), use DNA Star, primer premier software to design 2 primer fragments F 1 , F 2 Amplification of the avian IL-2 gene. Primers were synthesized by Dalian Bao Biological Engineering Company. Primer F1 5' end introduction Kpn I endonuclease site, F2 5' end introduces a flexible linker. The primer sequences are as follows:
[0070] F1: SEQ ID NO.1;
[0071] 5'-CGGGTACCATGTGCAAAGTACTGATCTTTGGC-3'
[0072] F2: SEQ ID NO.2;
[0073] 5'-GCTGCCGCCGCCGCCTTTTTGCAGATATCTCAC-3'
[0074]The NDV-I vaccine was diluted 50 times, inoculated into 10-day-old chicken embryos through the allantoic cavity, 0.2 mL / piece, and incubated at 37 °C. Undead chicken embryos were taken out 48-72 hours after inoculation, and the spleen was aseptically ground, and the total mRNA was extracted and amplified immediately according to the requiremen...
Embodiment 2
[0085] chIL-2-HN Construction of Fusion Gene Recombinant Yeast Expression Vector
[0086] use Kpn I. not I endonuclease was used to double-digest the PCR product of the above chIL-2-HN fusion gene and the Pichia pastoris expression vector pPICZαA, and put it in a water bath at 37 ℃ for 2 hours. The digested products were also identified by 1% agarose gel electrophoresis Finally, the gel recovery kit was used for recovery identification. The digested chIL-2-HN fusion gene and Pichia pastoris expression vector pPICZαA were connected overnight at 4°C at a molar ratio of 1:3. The ligated product was added to a polypropylene centrifuge tube containing 100 μL of competent DH5α, mixed gently, and then placed in an 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 placed in an ice bath for 2 min. Add 800 μL of LB medium preheated at 37 °C, and shake (100-150 rpm) at 37 °C for 45 ...
Embodiment 3
[0088] Construction of genetically engineered strains expressing chIL-2-HN fusion protein:
[0089] The positive recombinant yeast expression vector pPICZαA-chIL-2-HN obtained in Example 2 was linearized with SacI, and 5 μg of the linearized recombinant expression vector pPICZαA-chIL-2-HN was mixed with 80 μL of competent Pichia pastoris X- 33 phases were mixed, transferred to a pre-cooled 0.2 cm electric cup, placed on ice for 5 min, 1.5 kV, 25 μF, 200 Ω electric shock, immediately added 1 mL of pre-cooled 1 mol / L sorbitol, and 200 μL was spread on On a YPDS plate, cultivate at 30°C until a single colony appears; refer to the Pichia Expression Kit for detailed steps; use PCR to analyze Pichia transformants, use the boil-freeze-boil method to prepare PCR templates, and use primers F1 and F4: Reaction system Same as above, PCR reaction conditions: 94°C for 5 min; 94°C for 45 s, 48°C for 45 s, 72°C for 45 s, 25 cycles; 72°C for 6 min; identify and amplify a clone with a size of ...
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