Interferon alpha1b mutant and fusion protein containing interferon alpha1b and human serum albumin
A technology of human serum albumin and interferon α, which is applied in the field of interferon α1b mutants and their fusion proteins with human serum albumin, which can solve the problems of decreased biological activity of proteins
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Embodiment 1
[0047] Example 1: Construction of Engineering Bacteria Expressing Interferon α1b164 Serine Mutant and Confirmation of Expression Sequence
[0048] Extract the plasmid pET23b / IFN plasmid of interferon α1b as a template, and the primers of the reaction system are:
[0049] P1: 5'-TGCAGGAACGTCTGCGTAGTAAAGAATAACGAATTC-3',
[0050] P2: 5'-GAATTCGTTATTCTTACTACGCAGACGTTCCTGCA-3',
[0051] The reaction conditions were: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 53°C for 1 minute, extension at 72°C for 90 seconds, 30 cycles, and extension at 72°C for 5 minutes.
[0052] The PCR product was analyzed by agarose electrophoresis, and a DNA fragment of about 720bp was selected, digested with Nde I / EcoRI double enzymes, and the target DNA fragment of about 500bp was recovered by electrophoresis.
[0053] The pET-23b plasmid vector was digested with Nde I / EcoRI double enzymes, recovered by agarose electrophoresis and ligated with the target DNA f...
Embodiment 2
[0056] Example 2: Fermentation, purification and detection of interferon α1b163-site serine mutant
[0057] Construct the Escherichia coli engineered bacteria expressing the interferon α1b163 site serine mutant with the same method as in Example 1, then select a single bacterium colony to inoculate in the LB medium containing ampicillin after being activated by coating the plate (per liter uses peptone 10g, yeast powder 5g, NaCl10g, adjust the pH to 7.0), shake the flask at 37°C and 210 rpm to OD 600nm It is 0.6-0.8. Then inoculate 50L LB medium with 5% volume inoculum and carry out fermentation culture in 80L fermenter (containing 0.1% ampicillin per liter). Between -7.5, use the rotating speed to control the dissolved oxygen value between 4-8%. in OD 600nm After reaching 1.0, 10 g of IPTG was added according to the ratio of mass volume ratio 1:5000 to continue the induction culture for 3 hours, the induction culture temperature was 37 ° C, and LB medium was appropriately ...
Embodiment 3
[0063] Example 3: Fermentation, purification and detection of interferon α1b164-site serine mutant
[0064] The Escherichia coli engineered bacteria constructed by the method of Example 1 were activated and shake flask cultured according to the method of Example 2. After inoculating 140L of LB culture in a 200L fermenter with a volume inoculum of 6%, the fermentation culture was carried out, and the conditions were as in Example 2. After the induction culture time is over, put the tank at room temperature and centrifuge at 5000 rpm for 20 minutes to collect the bacteria, and the obtained bacteria are washed twice with the above-mentioned TE buffer to remove the main impurities in the fermentation broth.
[0065] Take 40 g of the thalli obtained from the above-mentioned treatment and add 480 ml of the above-mentioned TE buffer solution at a mass-to-volume ratio of 1:12, and add 4.8 g of lysozyme at a volume-to-mass ratio of 100:1 to treat it for more than 4 hours. Centrifuge f...
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