Gene synthesis duck alpha interferon gene and protein expression

Abstract

The invention uses the gene synthesis technology to obtain the duck alpha interferon gene and carry out pronucleus expression on interferon protein, belonging to the field of biological pharmacy. The invention comprises the following steps: carrying out codon reformation to the duck alpha interferon gene sequence, and combining the sequence by genes, building the pronucleus expression carrier containing the duck alpha interferon gene; converting escherichia coli by the pronucleus expression carrier containing the duck alpha interferon gene, carrying out induction expression, extracting protein, carrying out denaturation and renaturation, and researching antiviral activity. The interferon obtained by the test can resist the attack of vesicular disease viruses, and the specific activity reaches 5.6*103U / mg. The interferon produced by the method has simple technology, high expression amount and high bioactivity.

Description

a technical field

[0001] The invention belongs to the field of genetic engineering biopharmaceuticals, and relates to the prokaryotic expression of duck alpha interferon gene and interferon protein obtained by using the whole gene synthesis technology. Two background technology

[0002] At present, with the continuous expansion of the scale of duck raising in my country and the continuous improvement of the degree of intensification, the harm caused by duck viral diseases such as duck viral hepatitis, duck plague, and duck flu has become more and more serious, causing major damage to the duck industry. loss. The interferon system is an important defense system for the body to fight against viral infections. When the interferon system is activated, serum interferon can spread to various organs in the body within a few minutes, and it only takes a few minutes for cells to be exposed to interferon to produce antiviral State, moreover, animals can become resistant to superinfect...

Examples

Embodiment 1

[0046] Synthesis of embodiment 1 duck interferon alpha gene

[0047] Log in to GeneBank, search for the gene sequence encoding duck interferon alpha mature protein (serial number is AB128861), use SignalP 3.0Server software to predict that this gene may contain a signal peptide sequence, which is the first 30 amino acids of its amino acid sequence, use TargetP 1.1Server The software detects the function of this sequence and finds that it is necessary for the signal peptide that helps protein secretion rather than protein structure. Since duck interferon alpha is eukaryotic interferon, but follow-up research needs to be expressed in prokaryotes, eukaryotic signal The peptide cannot play a role in the prokaryotic cell environment due to the lack of corresponding receptors, so we removed the signal peptide sequence, leaving the gene sequence encoding the mature duck interferon alpha, which has a nucleotide length of 486bp; the duck interferon alpha According to the degeneracy of ...

Embodiment 2

[0049] Example 2 Prokaryotic expression of duck interferon alpha protein

[0050] 1. Transfer the correctly sequenced recombinant plasmid pET32a-DuIFN-α into BL21(DE3), pick a single colony for double enzyme digestion identification;

[0051] 2 Induced expression of positive recombinant bacteria

[0052] Pick a single colony containing the recombinant positive plasmid, inoculate it into LB liquid medium containing ampicillin at a concentration of 100 mg / mL, and cultivate overnight at 37°C with shaking. Inoculate the overnight cultured bacterial solution into Amp+ / LB liquid medium at a ratio of 1%, cultivate for 3 hours until the optical density of the bacterial solution reaches 0.4-0.6, immediately add IPTG until the working concentration reaches 1mM, continue to cultivate for 5 hours, and take every 1 hour 1ml bacterial solution, and carry out SDS-PAGE electrophoresis to determine the optimal induction time (see attached figure 1 );

[0053] 3 Soluble identification of duc...

Embodiment 3

[0057] Example 3 Extraction of Recombinant Duck Interferon Alpha

[0058] 1 Bacterial fragmentation

[0059]The induced bacteria were collected by centrifugation, washed three times with PBS, the cells were broken by ultrasonic, and the smear microscope was used to observe the breaking effect, and the supernatant was discarded to obtain crude inclusion bodies;

[0060] 2 Washing of inclusion bodies

[0061] Fully wash the inclusion body with the inclusion body washing solution containing 2M urea, centrifuge at 4°C, 12000rpm for 15min, and discard the supernatant;

[0062] 3 Denaturation of inclusion bodies

[0063] Resuspend the precipitate with inclusion body solution containing 8M urea, and dissolve and denature the inclusion body in a 30°C water bath, centrifuge to take the supernatant, and obtain the denatured protein solution;

[0064] 4 Target protein dialysis refolding

[0065] Add the denatured protein solution into the dialysis bag, put it into the PBS solution co...

a technical field

[0001] The invention belongs to the field of genetic engineering biopharmaceuticals, and relates to the prokaryotic expression of duck alpha interferon gene and interferon protein obtained by using the whole gene synthesis technology. Two background technology

[0002] At present, with the continuous expansion of the scale of duck raising in my country and the continuous improvement of the degree of intensification, the harm caused by duck viral diseases such as duck viral hepatitis, duck plague, and duck flu has become more and more serious, causing major damage to the duck industry. loss. The interferon system is an important defense system for the body to fight against viral infections. When the interferon system is activated, serum interferon can spread to various organs in the body within a few minutes, and it only takes a few minutes for cells to be exposed to interferon to produce antiviral State, moreover, animals can become resistant to superinfect...

Examples

Embodiment 1

[0046] Synthesis of embodiment 1 duck interferon alpha gene

[0047] Log in to GeneBank, search for the gene sequence encoding duck interferon alpha mature protein (serial number is AB128861), use SignalP 3.0Server software to predict that this gene may contain a signal peptide sequence, which is the first 30 amino acids of its amino acid sequence, use TargetP 1.1Server The software detects the function of this sequence and finds that it is necessary for the signal peptide that helps protein secretion rather than protein structure. Since duck interferon alpha is eukaryotic interferon, but follow-up research needs to be expressed in prokaryotes, eukaryotic signal The peptide cannot play a role in the prokaryotic cell environment due to the lack of corresponding receptors, so we removed the signal peptide sequence, leaving the gene sequence encoding the mature duck interferon alpha, which has a nucleotide length of 486bp; the duck interferon alpha According to the degeneracy of ...

Embodiment 2

[0049] Example 2 Prokaryotic expression of duck interferon alpha protein

[0050] 1. Transfer the correctly sequenced recombinant plasmid pET32a-DuIFN-α into BL21(DE3), pick a single colony for double enzyme digestion identification;

[0051] 2 Induced expression of positive recombinant bacteria

[0052] Pick a single colony containing the recombinant positive plasmid, inoculate it into LB liquid medium containing ampicillin at a concentration of 100 mg / mL, and cultivate overnight at 37°C with shaking. Inoculate the overnight cultured bacterial solution into Amp+ / LB liquid medium at a ratio of 1%, cultivate for 3 hours until the optical density of the bacterial solution reaches 0.4-0.6, immediately add IPTG until the working concentration reaches 1mM, continue to cultivate for 5 hours, and take every 1 hour 1ml bacterial solution, and carry out SDS-PAGE electrophoresis to determine the optimal induction time (see attached figure 1 );

[0053] 3 Soluble identification of duc...

Embodiment 3

[0057] Example 3 Extraction of Recombinant Duck Interferon Alpha

[0058] 1 Bacterial fragmentation

[0059]The induced bacteria were collected by centrifugation, washed three times with PBS, the cells were broken by ultrasonic, and the smear microscope was used to observe the breaking effect, and the supernatant was discarded to obtain crude inclusion bodies;

[0060] 2 Washing of inclusion bodies

[0061] Fully wash the inclusion body with the inclusion body washing solution containing 2M urea, centrifuge at 4°C, 12000rpm for 15min, and discard the supernatant;

[0062] 3 Denaturation of inclusion bodies

[0063] Resuspend the precipitate with inclusion body solution containing 8M urea, and dissolve and denature the inclusion body in a 30°C water bath, centrifuge to take the supernatant, and obtain the denatured protein solution;

[0064] 4 Target protein dialysis refolding

[0065] Add the denatured protein solution into the dialysis bag, put it into the PBS solution co...

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