Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF4 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this process is used by most people, but it has obvious disadvantages in the process of industrial application, which consumes a lot of manpower and material resources.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products