Construction and modification method of recombinant porcine long-acting-alpha interferon and preparation method of lyophilized injection

A technology for freeze-dried injection and alpha interferon, which is applied in the field of biopharmaceuticals to achieve the effects of improved half-life, high-efficiency expression and stable product quality

Active Publication Date: 2015-01-07
CHONGQING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no effective and practical drug for the treatment of porcine viral diseases, so the development of highly effective antiviral drugs is an important means for the treatment of porcine viral diseases, and it is important for the prevention and treatment of porcine viral infectious diseases and the development of pig production. necessary

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: A recombinant porcine long-acting-α interferon construction and PEG modification.

[0058] (1) Construction of recombinant porcine long-acting-α interferon:

[0059] 1. Primer design:

[0060] Using Primer 5.0, according to the porcine IFN-α gene sequence with accession number M28623.1 in GenBank, the primers for amplifying the porcine-α interferon mature peptide gene were designed. An EcoRI restriction site and an ATG initiation codon were added to the 5' end of the upstream primer, and an Xho I restriction site was added to the 5' end of the downstream primer.

[0061] The upstream primer is 5'-GGAATTCTGTGACCTGCCTCAG-3';

[0062] The downstream primer is 5'-CCGCTCGAGTCACTCCTTCTTCCTGAGT-3'.

[0063] Primers were synthesized by Takara Company.

[0064] 2. Extraction of porcine liver genomic DNA: the tissue / cell DNA (small amount) extraction kit (Watson Biotechnologies, Inc) was used to extract the porcine liver genomic DNA.

[0065] 2.1 Weigh about 20 m...

Embodiment 2

[0185] One, a kind of recombinant pig long-acting -α Interferon construction and PEG modification .

[0186] (1) Construction of recombinant porcine long-acting-α interferon:

[0187] 1. Primer design:

[0188] Using Primer 5.0, according to the porcine IFN-α gene sequence with accession number M28623.1 in GenBank, the primers for amplifying the porcine α-interferon mature peptide gene were designed. An EcoRI restriction site and an ATG initiation codon were added to the 5' end of the upstream primer, and an Xho I restriction site was added to the 5' end of the downstream primer.

[0189] The upstream primer is 5'-GGAATTCTGTGACCTGCCTCAG-3';

[0190] Downstream primer 5'-CCGCTCGAGTCACTCCTTCTTCCTGAGT-3'.

[0191] Primers were synthesized by Takara Company.

[0192] 2. Extraction of porcine liver genomic DNA: the tissue / cell DNA (small amount) extraction kit (Watson Biotechnologies, Inc) was used to extract the porcine liver genomic DNA.

[0193] 2.1 Weigh about 20 mg of...

experiment example 1

[0312] Experimental example 1 In vitro activity of recombinant porcine long-acting-α interferon:

[0313] 1. Dilute the recombinant porcine long-acting-α interferon sample with assay medium containing 7% newborn bovine serum to make the final protein concentration 10-100 μg / mL, and make 4-fold serial dilutions.

[0314] 2. Take the preserved virus, thaw at room temperature, and dilute to 100×CCID with serum-free DMEM medium 50 .

[0315] 3. Cultivate and plate the cells according to the above method, and cultivate until the cell abundance reaches more than 80%.

[0316] 4. Discard the supernatant, add 100 μL of serially diluted interferon to each well for 18 h, and add 7% assay medium to the blank control group and virus control group.

[0317] 5. Wash off the residual serum with a small amount of serum-free medium, add 100 μL of virus diluent to each well for challenge for 1 h, and add 100 μL of serum-free medium to the blank control group and the group with the highest con...

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Abstract

The invention provides a construction and modification method of recombinant porcine long-acting-alpha interferon and a preparation method of a lyophilized injection. Recombinant porcine-alpha interferon provided by the invention is prepared by adopting colon bacillus soluble expression, and the recombinant porcine long-acting-alpha interferon is obtained by using polyethylene glycol to perform modification according to the characteristics of polyethylene glycol, so that the half life of the recombinant porcine long-acting-alpha interferon in a pig body is prolonged to 44.5h and the porcine immunity is enhanced. The recombinant porcine long-acting-alpha interferon provided by the invention has excellent expression under high density and high volume of bacteria, production and purification processes are simple and novel, the cost is low, and the product yield and purity are improved. The lyophilized injection of the recombinant porcine long-acting-alpha interferon, provided by the invention, has the advantages of low cost, significant curative effect and stable product quality.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a method for constructing and modifying recombinant porcine long-acting-alpha interferon and a preparation method for freeze-dried injection thereof. Background technique [0002] Porcine interferon is one of the earliest animal interferons studied, and porcine interferon was reported in the 1980s. The porcine interferons that have been discovered include INF-α, β, ω, δ and INF-γ, and now the genes have been cloned, sequenced and mapped. In recent years, the induction conditions and physical and chemical activities of porcine interferon have been further studied, and a large number of clinical trials have been carried out on porcine leukocyte interferon. Studies have shown that it has a significant effect on many viral infectious diseases of pigs, such as epidemic diarrhea, swine fever, infectious gastroenteritis, etc., as well as bovine viral diarrhea, gosling plague...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C12N15/70C07K1/22C07K1/107A61K38/21A61K47/48A61K9/19A61P37/04A61P31/12
CPCA61K9/0019A61K9/19A61K38/00A61K47/60C07K14/56
Inventor 蔡家利吴胜昔王远强胡仁建户国达尹忠宝
Owner CHONGQING UNIV OF TECH
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