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Method for improving antiviral activity of recombinant swine interferon-alpha fusion protein

A porcine interferon and recombinant protein technology, applied in the field of interferon genetic engineering, can solve the problems of inability to guarantee the effective activity of drugs, high cost of use by farmers, and increased production costs, and achieve increased stability and activity, short production time, and easy production. The effect of purification

Inactive Publication Date: 2017-10-17
哈尔滨紫霞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, interferon was produced in the form of wild type. Although it is similar to the natural structure, its half-life is short, and the effective activity of the drug cannot be guaranteed.
The general method increases the half-life by linking the Fc fragment of the antibody, but due to the large molecular weight after linking the Fc, it cannot be expressed through the prokaryotic system, but can only be expressed through the eukaryotic expression system, which virtually increases the production cost, resulting in relatively high cost for farmers. high

Method used

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  • Method for improving antiviral activity of recombinant swine interferon-alpha fusion protein
  • Method for improving antiviral activity of recombinant swine interferon-alpha fusion protein
  • Method for improving antiviral activity of recombinant swine interferon-alpha fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of Recombinant Porcine Interferon-α Gene Plasmid

[0033] 1. Synthesize the required recombinant porcine interferon-α gene by chemical synthesis.

[0034] 2. Construction of recombinant porcine interferon-α gene and pET27b vector

[0035] The recombinant porcine interferon-α gene synthesized in the above step 1 was excised with restriction endonucleases BamHI and HindIII, connected with the pET27b vector cut by the same enzyme and transformed into Escherichia coli DH5ɑ competent, and ampicillin-resistant The recombinant porcine interferon-α gene was cloned into the pET27b vector after plasmid extraction, enzyme digestion identification, and sequencing, and the obtained recombinant plasmid was named pET-rIFN-α.

Embodiment 2

[0036] Example 2 Escherichia coli strain highly expressing recombinant porcine interferon-α gene E. coli Construction of Rosetta / pET-rIFN

[0037] Convert pET-rIFN-α to E. coli Rosetta, transformants were screened on LB plates containing ampicillin, and the obtained recombinants were proved by plasmid extraction, enzyme digestion identification, and sequencing analysis E. coli Rosetta / pET-rIFN-α was as expected.

Embodiment 3

[0038] Embodiment 3 Utilize Escherichia coli engineering bacteria E. coli Production of Recombinant Porcine Interferon-α by Rosetta / pET-rIFN-α

[0039] 1. Culture and fermentation of strains

[0040] Pick Escherichia coli Engineering Bacteria E. coli Rosetta / pET-rIFN-α, the engineered bacteria were inoculated in LB liquid medium according to the inoculum amount of 1%, cultured at a constant temperature of 37°C for 12-14 h, expanded at 1:100 the next day to an OD value of 0.4, and added IPTG to the final Concentration of 0.5mM, continue to cultivate for 3h, 4000r / min, centrifuge for 30min to collect bacteria.

[0041]2. Purification of recombinant porcine interferon-α

[0042] Ultrasonic crushing and centrifugation of the above-mentioned collected bacteria collected the supernatant, filtered the supernatant and purified it using the AKTA protein purification system, followed by affinity chromatography and molecular sieve chromatography to obtain purified recombinant porcin...

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Abstract

The invention discloses a method for improving antiviral activity of a recombinant swine interferon-alpha fusion protein. The nucleotide sequence of the fusion protein is of SEQ ID NO.1 as shown in the specification, and an amino acid sequence encoded by using the fusion protein is of SEQ ID NO.2 as shown in the specification. By adopting the method, a swine interferon-alpha gene and a swine antibody CH3 fragment are subjected to fusion expression, the stability of the protein is improved, the activity time is prolonged, and efficient preparation is achieved through a prokaryotic expression system, and tests show that the half-life period of the recombinant swine interferon-alpha expressed by using the protein is remarkably prolonged when being compared with that of natural swine interferon-alpha, and the bioactivity of the recombinant swine interferon-alpha is also prior to that of a natural product.

Description

technical field [0001] The present invention relates to a recombined gene, in particular to a recombinant porcine interferon-α gene. The present invention also relates to an expression vector containing the gene and an engineering strain. The present invention further relates to their use in preparing porcine interferon-α, which belongs to The field of interferon genetic engineering. Background technique [0002] In the past two years, due to the increasingly serious harm of livestock and poultry immunosuppressive diseases, immune enhancers have been widely used in livestock production. Animals are threatened by disease or in a state of stress, especially when livestock and poultry are immunosuppressed, the use of immune enhancers has a significant effect. With the advancement of science and technology and the needs of modern animal husbandry, more and more attention has been paid to the prevention and treatment of immune enhancers. Ideal immune enhancers with definite char...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/70C12N1/21C07K1/14C07K1/22C12R1/19
CPCC07K14/56C07K2319/21C07K2319/31C12N15/70
Inventor 吴云舟吴迪赵伟其他发明人请求不公开姓名
Owner 哈尔滨紫霞生物科技有限公司
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