Rapid purification method of gastric cancer targeted recombinant immunotoxin
A technology of immunotoxin and purification method, which is applied in the field of purification of gastric cancer-targeted recombinant immunotoxin, and can solve the problem of decreased affinity of active tumor cells
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Embodiment 1
[0062] Example 1 Identification of recombinant strain expressing rG17PE38KDEL
[0063] (1) Activation of the expression strain BL21(DE3)PLysS(pET-rG17PE38KDEL): Pick the glycerol-preserved bacterial solution with the inoculation loop and streak it on the solid LB(kana+) medium, incubate overnight in a 37°C incubator and pick a single colony, Inoculate in 5ml liquid LB (kana+) medium;
[0064] (2) Induced expression of the recombinant toxin rG17PE38KDEL: after inoculation into the liquid medium, shake culture at 180rpm / min for 6 hours at 37°C, and wait for the OD of the bacterial solution 600 When =0.4~0.6, add 1 mM inducer IPTG, 37°C, 180 rpm / min and continue to shake for 6 hours. Bacteria cultured under the same conditions without adding inducer IPTG were used as negative control.
[0065] (3) SDS-PAGE electrophoresis verification induction: Take 1 mL of bacterial liquid, add 5×SDS-PAGELoading Buffer after collecting the bacterial cells, bathe in boiling water for 10 min, a...
Embodiment 2
[0066] The western-blot identification of embodiment 2 recombinant protein
[0067] Prepare a 10% SDS-PAGE electrophoresis gel, add 10 μL of the induced protein sample prepared in Example 1 to each well, and perform SDS-PAGE electrophoresis; Color development, final chemiluminescence imaging system analysis and photographing (see figure 2 ).
Embodiment 3
[0068] Embodiment 3 expands the culture expression condition optimization, the result sees image 3
[0069] (1) Optimization of induction temperature
[0070] Inoculate BL21(DE3)PLysS(pET-rG17PE38KDEL) cultured by shaking overnight into 1LLB liquid medium (kana+) with 1% inoculum, and cultivate to OD at 180rpm / min at 37℃ 600 0.4, insert IPTG with a final concentration of 1.0 mM, and continue culturing at 180 rpm for 6 hours at 16°C, 20°C, 35°C, 30°C, and 37°C, respectively.
[0071] (2) Optimization of induction timing
[0072] Inoculate BL21(DE3)PLysS(pET-rG17PE38KDEL) cultured by shaking overnight into 1LLB liquid medium (kana+) with 1% inoculum, culture at 180rpm / min 37℃ for 0.5h, 1.0h, 1.5h, 2h , 2.5h, and 3.0h, insert IPTG with a final concentration of 1.0mM, and continue culturing at 180rpm at 37°C for 6h.
[0073] (3) Optimization of inducer dosage
[0074]The BL21(DE3)PLysS (pET-rG17PE38KDEL) cultured by shaking overnight was inoculated into 1 LLB liquid medium (...
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