A kind of Bacillus enteromorpha y15-8 and its anti-tumor activity protein
A technology with anti-tumor activity and Bacillus, which is applied in the field of marine biological resources and biomedicine, can solve the problems of low overall utilization rate, insufficient excavation and utilization of microorganisms, and unrealized large-scale utilization, achieving good research value and The effect of applying the foreground
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example 1
[0033] The screening isolation process of example 1 Bacillus enteromorpha Y15-8
[0034] (1) Primary screening of activity
[0035] Fresh Enteromorpha was taken from the sea area of Qingdao, ground and pulverized into puree, and the Enteromorpha samples were selectively coated with different media such as seawater basal medium 2216E, LB medium and beef extract peptone medium NRG. , Streak and separate culture, a total of 27 single colonies were obtained. The anti-tumor activity of the fermentation products of these 27 strains of Enteromorpha symbiotic microorganisms was screened, and it was found that 2 strains had an inhibition rate of more than 50% on liver cancer cell BEL-7402.
[0036] (2) Strain re-screening
[0037] The proliferation inhibitory effect of the above two active strains on other tumor cells, including human liver cancer cell HepG2, human lung adenocarcinoma cell A549 and human lung cancer cell NCI-H460, was determined. The results showed that the fermen...
example 3
[0048] The fermentation culture of example 3 Bacillus enteromorpha Y15-8
[0049] The screened Bacillus enteromorpha Y15-8 (the Latin name is Bacillus sp.Y15-8, was deposited in the China Center for Type Culture Collection on December 12, 2016, the preservation address: Wuhan University, Wuhan, China, and the preservation number is CCTCC M2016744. Connected in a test tube containing 4mL seed culture solution, seed culture solution: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, the above is the final concentration of each component, pH7.5. Place at 30°C, 200rmp Cultivate overnight in a constant temperature shaking incubator; make a gradient dilution coating plate, solid medium: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, the above are the final concentrations of each component, pH 7.5 .Put the coated plate in a 30°C constant temperature incubator and culture it upside down for 18-36h; pick a single colony and inoculate it in a 4mL test tube seed culture solution, an...
example 4
[0050] The preparation of example 4 Bacillus enteromorpha Y15-8 active protein
[0051] (1) ultrafiltration
[0052] 8 L of the collected fermentation supernatant was concentrated by ultrafiltration. Then, ultrafiltration is performed with ultrafiltration membranes with a molecular weight of 3kDa and 50kDa to obtain an active component with a molecular weight of 3-50kDa.
[0053] (2) Ammonium sulfate precipitation
[0054] Slowly add ammonium sulfate powder to the 3-50kDa component, stir gently to make the concentration reach 60% (mass percentage concentration), control the temperature at 4°C during the period, and leave it in the refrigerator for 10-12h after all dissolve; at 9800rpm , centrifuge under the condition of 25min, collect the supernatant, continue to add ammonium sulfate powder, make its concentration reach 75% (mass percentage concentration), during the control temperature 4 ℃, after all dissolving, the refrigerator stands still for 10~12h; At 9800rpm, 25min C...
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