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Recombinant canine alpha-interferon optimized gene, strain and efficient expression

A canine interferon, recombinant vector technology, applied in interferon, cytokine/lymphokine/interferon, genetic engineering and other directions, can solve the problems of low specific activity and low expression rate of recombinant canine interferon alpha

Active Publication Date: 2011-10-19
中科拜克(天津)生物药业有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, recombinant canine interferon α has the characteristics of low expression rate and low specific activity, usually the expression level will not exceed 30%

Method used

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  • Recombinant canine alpha-interferon optimized gene, strain and efficient expression
  • Recombinant canine alpha-interferon optimized gene, strain and efficient expression
  • Recombinant canine alpha-interferon optimized gene, strain and efficient expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, the construction of engineering bacteria

[0025] 1. Acquisition of canine alpha interferon gene

[0026] Referring to the amino acid sequence of canine α-interferon in Genbank M28624, combined with the reported Escherichia coli preferred codons, 30 nucleotide sequences encoding canine α-interferon were artificially synthesized, constructed into sequencing vectors, and sequenced for verification.

[0027] 2. Construction of recombinant expression vectors

[0028] The target gene fragment was recovered by restriction endonuclease EcoRI / SalI double digestion; the prokaryotic expression vector pBV220 was recovered by restriction endonuclease EcoRI / SalI double digestion; the target vector fragment was recovered; ligated and transformed into E. coli E.Coli.BL21( DE3) Competent cells, positive clones were screened by PCR, and then verified by sequencing to obtain a recombinant expression plasmid pBV220 / CaIFN-α with a completely correct sequence (see figure 1 ...

Embodiment 2

[0032] Embodiment 2, the production process of preparing canine interferon alpha with recombinant bacteria of the present invention

[0033] 1. Fermentation expression

[0034] Temperature-induced expression: Inoculate the recombinant bacteria in 500ml of TB culture solution according to the inoculum amount of 1%, and shake at 220rpm at 30°C for 30-36h. The primary bacteria were inoculated at 5% in a 15-liter fermenter, cultured at 30°C until OD=5-6, then rapidly raised to 42°C, and induced for 3 hours. The expression products were detected by SDS-PAGE electrophoresis, such as Figure 4 As shown, the expression level of the target protein accounts for about 40% of the total protein of the bacteria, and the yield is extremely high.

[0035] 2. Broken

[0036] After the induction, centrifuge at 5000r / min to collect the bacteria. Use a high-pressure homogenizer to crush the bacteria, centrifuge at 8000r / min for 20min, and collect the precipitate, which is the inclusion body. ...

Embodiment 3

[0041] Example 3 Determination of Antiviral Specific Activity of Recombinant Canine Interferon-alpha

[0042] Micro cytopathic inhibition method

[0043] MDCK was inoculated into a 96-well plate, and after growing into a single layer, the growth medium was discarded, and 10-fold dilution of recombinant canine interferon α was added, and each dilution was repeated 8 times. After 24 hours of culture, the supernatant was discarded, and vesicles with 10TCID50 Stomatitis virus (VSV) was used for poisoning, colleagues set up negative control (only add 10 times diluted interferon, no virus), positive control (only add virus, no interferon), blank control (no interferon , without adding virus), observe the cell lesions day by day under an inverted microscope, and finally stain the cells with crystal violet staining solution, measure the OD550 value of the sample on each instrument, and calculate the dilution equivalent to 50% of the OD value of the lesion according to the exponential ...

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Abstract

The invention relates to an optimized sequence of a recombinant canine alpha-interferon coding gene, an expression vector containing the optimized sequence and a recombinant canine alpha-interferon Escherichia coli strain containing the expression vector. The strain provided by the invention can efficiently express the canine alpha-interferon, the expression amount can reach higher than 40%, and the specific activity of the expressed protein is high. The invention also relates to a method for preparing the canine alpha-interferon.

Description

technical field [0001] The invention relates to an optimized recombinant canine alpha interferon gene and an expression strain, belonging to the field of genetic engineering biopharmaceuticals. Background technique [0002] Canines, as companion animals, police dogs, rescue dogs, etc., occupy an increasingly important position in human life, and various viral diseases of dogs that accompany it also occur repeatedly in people's lives, and some of them have become zoonotic diseases. Pathogens have aroused the attention of the whole society. However, in terms of veterinary clinical practice, there is currently no specific drug for the treatment of viral diseases. [0003] In 1957, British scientists Alick Isaacs and Iean Lindenmann first discovered a substance that can interfere with virus reproduction, called interferon (interferon, IFN), and began the research work on interferon. [0004] In 1987, Himmler et al first started the research work on canine interferon α (CaINF-α...

Claims

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Application Information

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IPC IPC(8): C12N15/21C12N15/70C12N1/21C12P21/00C07K14/56C07K1/18C12R1/19
Inventor 陈黄实陈嫚毕清贵毕清华史晓海
Owner 中科拜克(天津)生物药业有限公司
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