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

Active Publication Date: 2014-04-02
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

At present, this process is used by most people, but it has obvious disadvantages in the p

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  • Preparation method of recombinant pig alpha interferon
  • Preparation method of recombinant pig alpha interferon
  • Preparation method of recombinant pig alpha interferon

Examples

Experimental program
Comparison scheme
Effect test

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

[0...

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Abstract

The invention constructs and screens a recombinant pichia pastoris strain PoIFN-alpha/GS115 which can efficiently perform secretory expression of pig alpha interferon, overcoming the problems of complex purification process and low biology activity and the like when a recombinant interferon is expressed and prepared by using escherichia coli. According to the invention, a specific fermenting technology which is different from a traditional high-density fermenting technology and is suitable for efficient expression of a target gene is established. The fermenting technology is short in production cycle, is simple in purification process and is low in cost.

Description

technical field [0001] The invention relates to a preparation method of recombinant porcine alpha interferon. Background technique [0002] Porcine interferon-α (porcine interferon-α, PoIFN-α) is produced by porcine white blood cells. About 17 gene copies are distributed on chromosome 1, and at least 20 IFN-α subtypes have been translated. So far, only some of them have been cloned. Sequencing and expression. Because PoIFN-α has good antiviral activity, people have been trying to use genetic engineering to clone and express recombinant porcine interferon-α (rPoIFN-α), so that it has the natural PoIFN-α Broad-spectrum, highly effective antiviral activity. [0003] IFN-α is a biological protein that can efficiently induce cells to produce antiviral protein (AVP) and other effector molecules. AVP is a type of enzyme with no specific effect and has a certain inhibitory effect on most viruses. IFN-α can not only interrupt the virus replication process of infected cells in the ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12P21/02C12R1/84
Inventor 王一成李宝臣袁秀芳蓝胜芝张存李军星徐丽华
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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