A kind of il-1β recombinant protein of scorpionfish xu's and its preparation method and application
A recombinant protein and protein-binding technology, applied in the field of genetic engineering, can solve the problems of adverse effects of proteins, long time, cumbersome operation of denaturation and renaturation of inclusion bodies, etc.
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Embodiment 1
[0036] Embodiment 1: The construction of the expression vector pET-HMT-IL1B of the flat scorpionfish IL-1β of Xu's
[0037] Using the cDNA of the head kidney of Scorpius xushi as a template, the IL of scorpionfish IL was amplified by PCR using primers O-il1b-F / R (Shenggong, Shanghai) and reagent 2×PhantaMax Master Mix (Novizant, Nanjing) The DNA sequence of the open reading frame (ORF) of the -1β gene (il1b) ( figure 1 Middle B). It was ligated into pCE2 TA / Blunt Zero vector (Novazyme, Nanjing) by TOPO ligation to transfect DH5α Escherichia coli, a single colony was screened on ampicillin solid medium, and Sanger sequencing and NCBI BLAST were used to identify whether the sequence was correct. Using the above-mentioned il1b monoclonal bacterium solution of Scorpius xushii as a template, PCR amplification was performed using primers PE-il1b-F / R and 2×Phanta MaxMaster Mix to obtain a double-stranded DNA fragment corresponding to the mature peptide of il1b with homology arms ( ...
Embodiment 2
[0042] Embodiment 2: Preparation of the expression strain of Escherichia coli IL-1β of Scorpio xushi
[0043] The expression vector pET-HMT-IL1B was transfected into Rosetta B(DE3) competent Escherichia coli (Ang Yu, Shanghai), and cultured with ampicillin-chloramphenicol double antibody LB solid medium at 37°C overnight to screen a single colony, namely Xu's The prokaryotic expression engineering strain pET-HMT-IL1B-RosettaB(DE3)( Figure 5 Middle E). Pick a single colony and culture it overnight in 10mL ampicillin-chloramphenicol double-antibody LB liquid medium at 37°C at 220 rpm, then inoculate it in 100mL double-antibody TB liquid medium at a ratio of 1:100 at 37°C at 220 Cultivate at RPM to OD600 to 0.6-0.8, then add IPTG to make the final concentration 0.1mM (take out 50mL bacterial liquid as the control before induction), 20℃, 120 RPM for 12h, and then centrifuge at 4000g for 10min to collect the bacteria. Discard the supernatant, resuspend the bacteria in 5 mL of no...
Embodiment 3
[0044] Example 3: Soluble Expression, Protein Purification and Tag Removal of IL-1β Recombinant Protein from Scorpionfish Xu's
[0045] Inoculate in 500mL double-antibody TB liquid medium at a ratio of 1:100, culture at 37°C, 220 rpm until OD600 reaches 0.6-0.8, then add IPTG to make the final concentration 0.1mM, 20°C, 120 rpm After culturing for 12 hours, collect the cells by centrifugation at 4000g for 10 minutes, discard the supernatant, resuspend the cells in 50 mL of non-denaturing lysis buffer, add lysozyme at a final concentration of 1 mg / mL, lyse on ice for 30 minutes, ultrasonically break at 60 W for 15 minutes, supersonicate for 5 seconds and stop for 5 seconds , centrifuge at 12000g for 10min to discard the precipitate, retain the supernatant, filter with a 0.45 μm or 0.22 μm filter membrane to remove insoluble particles, use NTA-Ni filler (Abbison, Shanghai) for His tag affinity purification, and obtain a purity > 90% His6-MBP fusion IL-1β recombinant protein ( ...
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