Preparation method of recombinant pig alpha interferon
A technology of α-interferon and recombinant plasmid, which is applied in the field of preparation of recombinant porcine α-interferon, can solve the problems of consuming manpower and material resources, and achieve the effects of short production cycle, simplified purification process, and shortened production cycle
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Embodiment 1
[0031] Embodiment 1 Transformation and artificial synthesis of porcine alpha interferon gene
[0032] According to the determined porcine interferon-alpha gene sequence and the multiple cloning site (MCS) sequence of the expression vector pPIC9K, the porcine interferon-alpha gene sequence was artificially synthesized. The synthetic porcine alpha interferon gene sequence is dominated by yeast preferred codons. Its sequence is shown in SEQ ID No.1.
Embodiment 2
[0033] Example 2 Construction and Identification of Recombinant Plasmid pPIC9K-PoIFN-α
[0034] 1. Construction of recombinant plasmids
[0035] Using the artificially synthesized porcine interferon-α gene as a template, using IFN-α-P1 and IFN-α-P2 as primers, and using Pfu DNA polymerase to amplify the porcine interferon-α gene, the DNA product is without T at both ends The blunt end of the PCR product was digested with EcoRI to make the 3' sticky end. The expression vector pPIC9K was digested with SnaB1 / EcoRI double enzymes, and the 5' end was also formed as a blunt end, while the 3' end was formed as a sticky end. The digested porcine interferon alpha fragment and the expression vector pPIC9K were subjected to agarose gel electrophoresis, and the gel was recovered and purified.
[0036] Wherein, the primer IFN-α-P1 sequence is shown in SEQ ID No.2; the primer IFN-α-P2 sequence is shown in SEQ ID No.3.
[0037] Ligate the porcine α-interferon gene fragment with the expres...
Embodiment 3
[0047] Embodiment 3 fermentation culture
[0048] (1) Seed cultivation
[0049] 1. First-class seeds: Take PoIFN-α / GS115 bacteria stored at -80°C, inoculate at a ratio of 1:250, inoculate about 200 μl into a 500mL Erlenmeyer flask with 50ml YPD, and culture overnight at 220r / min at 30°C with vibration;
[0050] 2. Second-level seeds: Take the first-level seed liquid, inoculate it in BMGY culture medium at a ratio of 1:100-1:200, and cultivate it at 30°C and 220 rpm for 24 hours until the OD600 reaches about 25-30.
[0051] (2) Tank culture and induced expression
[0052] 1. Vaccination
[0053] Put all the secondary seeds into the tank at a ratio of 5%-10%, set the dissolved oxygen to 30%, and connect dissolved oxygen, stirring, and ventilation.
[0054] The fermentation expression medium used in this stage is the improved BMMY medium. The formula of the improved 1LBMMY is as follows:
[0055] 1% yeast extract (Yeast extract) 10g
[0056] 2% tryptone (Tryptone) 20g
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