Gene synthesis duck alpha interferon gene and protein expression
An alpha interferon, whole gene technology, applied in the direction of interferon, cytokine/lymphokine/interferon, genetic engineering, etc., can solve problems such as loss of duck farming
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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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