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Method for promoting antiviral activity of recombined dog interferon-gamma fusion protein

A canine interferon and recombinant protein technology, applied in the field of interferon genetic engineering, can solve the problems of high use cost and short half-life for farmers, and achieve the effects of improving antiviral activity, large expression amount and short production time

Inactive Publication Date: 2017-09-19
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-γ was produced in the form of wild type. Although it is similar to the natural structure, its half-life is short, resulting in high cost for farmers.

Method used

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  • Method for promoting antiviral activity of recombined dog interferon-gamma fusion protein
  • Method for promoting antiviral activity of recombined dog interferon-gamma fusion protein
  • Method for promoting antiviral activity of recombined dog interferon-gamma fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of recombinant canine interferon-γ gene plasmid

[0033] 1. Synthesize the required recombinant canine interferon-γ gene by chemical synthesis.

[0034] 2. Construction of recombinant plasmid expressing recombinant canine interferon-γ gene.

[0035] The recombinant canine interferon-γ gene synthesized in the above step 1 was digested with restriction endonucleases BamHI and HindIII, connected with the pET27b vector after being cut by the same enzymes and transformed into Escherichia coli DH5α competent, and screened for ampicillin resistance. The recombinant canine interferon-γ gene was cloned into the pET27b vector after plasmid extraction, enzyme digestion identification, and sequencing, and the resulting recombinant plasmid was named pET-rCaIFN-γ.

Embodiment 2

[0036] Example 2 Escherichia coli strain highly expressing recombinant canine interferon-γ gene E. coli Construction of Rosetta / pET-rCaIFN-γ

[0037] Convert pET-rCaIFN-γ 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-rCaIFN-γ was as expected.

Embodiment 3

[0038] Embodiment 3 Utilize Escherichia coli engineering bacteria E. coli Rosetta / pET-rCaIFN-γ Production of Recombinant Canine Interferon-γ

[0039] 1. Culture and fermentation of strains

[0040] Pick Escherichia coli Engineering Bacteria E. coli Rosetta / pET-rCaIFN-γ, inoculate engineering bacteria in LB liquid medium according to 1% inoculation amount, culture at 37°C for 12-14 h, expand culture at 1:100 the next day to OD value of 0.4, add IPTG to the final Concentration 0.5 mM, continue to cultivate for 3 hours, centrifuge at 4000 r / min for 30 minutes to collect bacteria.

[0041] 2. Purification of recombinant canine interferon-γ

[0042]Ultrasonic crushing and centrifugation of the above-mentioned collected bacteria was followed by supernatant collection, the supernatant was filtered and then purified by the AKTA protein purification system, and affinity chromatography and molecular sieve chromatography were performed successively to obtain purified recombinant c...

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Abstract

The invention discloses a preparation method for recombined dog interferon-gamma fusion protein. The nucleotide sequence thereof is shown as SEQ ID NO.1. The encoded protein amino acid sequence is shown as SEQ ID NO.2. According to the invention, the fusion expression of a dog interferon-gamma gene and a dog immune globulin CH3 fragment is adopted, so that the increasing of protein stability and activity is benefited and the protein expression quantity is increased. An experiment proves that the biological activity of the recombined dog interferon-gamma expressed according to the invention is obviously higher than the biological activity of natural dog interferon-gamma.

Description

technical field [0001] The present invention relates to a rearranged gene, in particular to a recombinant canine interferon-γ gene, the present invention also relates to an expression vector containing the gene and an engineering strain, and the present invention further relates to their use in preparing canine interferon-γ, It belongs to the field of interferon genetic engineering. Background technique [0002] Dogs, as human companion animals, play an increasingly important role in human life. Various canine viral diseases accompanying it also occur repeatedly in people's lives, and some of them become zoonotic pathogens. However, in terms of veterinary clinical practice, there is currently no specific drug for the treatment of viral diseases. With the advancement of science and technology and the needs of the modern veterinary industry, more and more attention has been paid to the prevention and treatment of immune enhancers. Ideal immune enhancers with definite characte...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/70C12N1/21C07K1/22C07K1/16
CPCC07K14/57C07K2319/30C07K2319/21
Inventor 朱升龙吴云舟朱明杰赵伟何国栋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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