Method for recombinant expression of human leukemia inhibitory factor
A technology for inhibiting factors and leukemia, applied in the fields of molecular biology and genetic engineering, can solve the problems of high hydrophobicity, difficulty in recombinant expression, etc., and achieve the effect of low production cost
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Embodiment 1
[0042] Construction of expression vector for fusion protein of luciferase and leukemia inhibitory factor
[0043] Using pNL1.2 (Promega, Madison, WI, USA) as a template, PCR was amplified, restriction sites NdeI and EcoRI were introduced, pET vector was ligated, and the ligated product was transformed into Escherichia coli DH12S strain (purchased from Invitrogen). Pick several transformed clones to extract plasmids, and check whether the target gene is correctly inserted by restriction endonuclease digestion. Select the plasmid with the target gene for DNA sequence determination, and select the correct DNA sequence for sequencing. The pNLuc with 6×His-NanoLuc-tag at the N-terminal was obtained. Digested with EcoRI and HindIII.
[0044] The leukemia inhibitory factor with EcoRI and HindIII restriction sites at both ends was digested and connected to pNLuc which was digested with EcoRI and HindIII in advance. The ligation product was transformed into Escherichia coli DH12S st...
Embodiment 2
[0046] Induced Expression of Fusion Protein of Luciferase and Leukemia Inhibitor
[0047] Transform the E. coli expression strain Rosetta-gami2 (DE3) (Merck) with the pNLuc / LIF plasmid constructed in Example 1, and the transformed clone grows in a medium containing ampicillin sodium (100 μg / ml), chloramphenicol (25 μg / ml), tetracycline hydrochloride (10 μg / ml) on solid LB medium. Pick a single clone and inoculate it in 50 ml liquid LB medium (containing 100 μg / ml ampicillin sodium, 25 μg / ml chloramphenicol, 10 μg / ml tetracycline hydrochloride), and culture overnight at 37° C. with shaking (250 rpm). Inoculate 20ml of the above bacterial solution into 1000ml liquid LB medium (containing 100μg / ml ampicillin sodium, 25μg / ml chloramphenicol, 10μg / ml tetracycline hydrochloride), and culture at 37°C with shaking (rotating speed 250rpm) until the bacterial solution is at 600nm The absorbance of the solution was about 1.0, and then IPTG was added to a final concentration of 1 mmol / l....
Embodiment 3
[0049] Separation and Purification of Fusion Protein of Luciferase and Leukemia Inhibitor
[0050] Centrifuge (5000g, 5min) 1000ml of the bacterium solution induced by Example 2, remove the supernatant, and wash the thalline with 60ml lysis buffer (20mM Tris-Cl (pH8.5), 100mM NaCl). The resuspended bacteria were disrupted on ice by ultrasonic (power 200W, ultrasonic 5s, intermittent 3s, total ultrasonic time 30min). The crushed bacterial liquid was centrifuged (12000g, 30min), and the supernatant was taken, which contained recombinant leukemia inhibitory factor. The supernatant was filtered and loaded onto a metal chelate (Ni 2+ ) on the chromatography column with a flow rate of 1ml / min. Then rinse with a lysis buffer containing 30mM imidazole, and finally elute the leukemia inhibitory factor with a lysis buffer containing 250mM imidazole, and collect the elution peaks. According to the identification result of SDS-PAGE, the recovery rate of the recombinant leukemia inhibit...
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