Preparation method of panda interferon
A giant panda, prokaryotic expression technology, applied in the field of protein engineering bacteria expression and preparation
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Embodiment 1
[0017] Example 1: Construction of the prokaryotic expression method of giant panda IFN-α3
[0018] Materials and Methods
[0019] 1. Genes, strains and vectors
[0020] The whole gene of giant panda IFN-α3 was synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the prokaryotic expression vector pET32a and strain RosettatagamiB (DE3) were purchased from Shanghai Sangon Bioengineering Co., Ltd.
[0021] 2. Construction of expression vector
[0022] According to the giant panda IFN-α type 3 gene sequence (DQ392970) in GeneBank, after sequence optimization, the signal peptide was removed and BamHI and XhoI restriction sites were added to synthesize the giant panda type 3 IFN-α gene 495bp without the signal peptide, which was sequenced After confirmation, clone into pET32a expression vector, completed by Shanghai Sangon Bioengineering Co., Ltd.
[0023] 3. Induced expression of recombinant proteins
[0024] After activating the expression bacteria, cultivate to OD 600...
Embodiment 2
[0027] Example 2: The method for measuring the activity of giant panda IFN-α3 expressed in prokaryotes
[0028] VSV-WISH detection system
[0029] Make WITH cells no less than 1X10 with 1640 culture medium containing 10% fetal bovine serum 5 cells / ml of cell suspension, add the cell suspension to a 96-well cell culture plate, 200 μl per well. Put in 5% CO 2 Incubate at 37°C for about 48 hours in an incubator until a uniform cell monolayer is formed. Then, using recombinant human interferon α2b for injection (1 million IU / ml) as a standard, the purified and determined recombinant giant panda IFN-α3 and human interferon were diluted 100 times with 1640 culture medium and then serially diluted 2 times. , add 50 μl / well into 96-well cell culture plate, put in 5% CO 2 Continue to incubate overnight at 37°C in the incubator, and then inoculate 100TCID assayed by WISH cells 50 VSV, 50 μl / well per well, set in 5% CO 2 Incubate at 37°C in an incubator, set up blank control (cells...
Embodiment 3
[0034] Example 3: Prokaryotic expression results of giant panda IFN-α3
[0035] The pET32a-melIFN-α3 recombinant plasmid was obtained, and the size of the double restriction fragment was consistent with the size of the whole gene of giant panda α3 type, and the sequence was confirmed to be correct by sequencing. The recombinant plasmid was transformed into RosettatagamiB(DE3) competent cells to obtain engineering bacteria RmelIFN-α3( figure 1 ).
[0036] The results of SDS-PAGE electrophoresis showed that RmelIFN-α3 had a better effect of inducing overnight expression at 20°C ( figure 2 ). Purification and detection of the target protein, the results show that the detection of the target protein was obtained with a purity of more than 90%.
[0037] Determination of the concentration of giant panda melIFN-α3, the results are shown in Table 1
[0038] The recombinant protein concentration determined in Table 1 was 1.66 mg / ml.
[0039]
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